Compounds and methods for modulating smn2

ABSTRACT

Provided are compounds, methods, and pharmaceutical compositions for modulating SMN2 RNA and/or protein in a cell or subject. Such compounds, methods, and pharmaceutical compositions are useful to ameliorate at least one symptom of a neurodegenerative disorder. Such symptoms include reduced muscle strength; inability or reduced ability to sit upright, to stand, and/or walk; reduced neuromuscular activity; reduced electrical activity in one or more muscles; reduced respiration; inability or reduced ability to eat, drink, and/or breathe without assistance; loss of weight or reduced weight gain; and/or decreased survival.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0367USSEQ_ST25.txt, created on Feb. 26, 2021, which is 44 KB insize. The information in the electronic format of the sequence listingis incorporated herein by reference in its entirety.

FIELD

Provided are compounds, methods, and pharmaceutical compositions formodulating SMN2 RNA in a cell or subject. Such compounds, methods, andpharmaceutical compositions are useful to ameliorate at least onesymptom of a neurodegenerative disorder. Such symptoms include reducedmuscle strength; inability or reduced ability to sit upright, to stand,and/or walk; reduced neuromuscular activity; reduced electrical activityin one or more muscles; reduced respiration; inability or reducedability to eat, drink, and/or breathe without assistance; loss of weightor reduced weight gain; and/or decreased survival.

BACKGROUND

Proximal spinal muscular atrophy (SMA) is a genetic neurodegenerativedisorder characterized by the loss of spinal motor neurons. SMA is anautosomal recessive disease of early onset and is a leading geneticcause of death among infants. The severity of SMA varies among patientsand it has thus been classified into four types. Type I SMA is the mostsevere form with onset at birth or within 6 months, and typicallyresults in death within 2 years. Children with Type I SMA are unable tosit or walk. Type II SMA is the intermediate form and patients are ableto sit, but cannot stand or walk. Patients with Type III SMA, a chronicform of the disease, typically develop SMA after 18 months of age(Lefebvre et al., Hum. Mol. Genet., 1998, 7, 1531-1536). Type IV SMA isa milder form and typically has an onset after 18 years of age,sometimes after 10 years of age; patients with Type IV SMA experiencelimited mild motor impairment, are able to walk in adulthood andgenerally do not have respiratory or nutritional problems (Farrar etal., Ann. Neurol., 2017, 81, 355-368; D′Amico et al., Orphanet J. ofRare Diseases, 2011, 6:71).

The molecular basis of SMA is the loss of both copies of survival motorneuron gene 1 (SMN1), which may also be known as SMN Telomeric, andencodes a protein that is part of a multi-protein complex thought to beinvolved in snRNP biogenesis and recycling. A nearly identical gene,SMN2, which may also be known as SMN Centromeric, exists in a duplicatedregion on chromosome 5q13 and modulates disease severity. Although SMN1and SMN2 have the potential to code for the same protein, expression ofthe normal SMN1 gene results solely in expression of full-lengthsurvival motor neuron (SMN) protein, while expression of the SMN2 generesults in two different protein forms, full-length SMN2 protein, and atruncated SMN2 protein, SMNA7 protein. SMN2 contains a translationallysilent mutation at position+6 of exon 7, which results in inefficientinclusion of exon 7 in SMN2 transcripts. Thus, the predominant form ofSMN2 is a truncated version, lacking exon 7, which is unstable andinactive (Cartegni and Krainer, Nat. Genet., 2002, 30, 377-384).Expression of the SMN2 gene results in approximately 10-20% of thefull-length SMN protein and 80-90% of the unstable/non-functional SMNA7protein. SMN protein plays a well-established role in assembly of thespliceosome and may also mediate mRNA trafficking in the axon and nerveterminus of neurons.

It is an object herein to provide compounds, methods, and pharmaceuticalcompositions for the treatment of SMA.

SUMMARY OF THE INVENTION

Provided herein are compounds, methods, and pharmaceutical compositionsfor modulating splicing of SMN2 RNA in a cell or subject. In certainembodiments, compounds useful for modulating splicing of SMN2 RNA areoligomeric compounds. In certain embodiments, oligomeric compoundsincrease the amount of SMN2 RNA including exon 7. In certainembodiments, oligomeric compounds increase full-length SMN2 proteinexpression. In certain embodiments, the oligomeric compound comprises amodified oligonucleotide. In certain embodiments, the subject has aneurodegenerative disease. In certain embodiments, the subject hasSpinal Muscular Atrophy (SMA).

Also provided are methods useful for ameliorating at least one symptomof a neurodegenerative disease. In certain embodiments, theneurodegenerative disease is SMA. In certain embodiments, symptomsinclude reduced muscle strength; inability or reduced ability to situpright, to stand, and/or walk; reduced neuromuscular activity; reducedelectrical activity in one or more muscles; reduced respiration;inability or reduced ability to eat, drink, and/or breathe withoutassistance; loss of weight or reduced weight gain; and/or decreasedsurvival. In certain embodiments, provided herein are modifiedoligonucleotides for treating SMA.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive. Herein, the use of the singular includes theplural unless specifically stated otherwise. As used herein, the use of“or” means “and/or” unless stated otherwise. Furthermore, the use of theterm “including” as well as other forms, such as “includes” and“included”, is not limiting. Also, terms such as “element” or“component” encompass both elements and components comprising one unitand elements and components that comprise more than one subunit, unlessspecifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, and GenBank and NCBI reference sequence recordsare hereby expressly incorporated-by-reference for the portions of thedocument discussed herein, as well as in their entirety.

Definitions

Unless specific definitions are provided, the nomenclature used inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well-known and commonly used in theart. Where permitted, all patents, applications, published applicationsand other publications and other data referred to throughout in thedisclosure are incorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

As used herein, “2′-deoxyribonucleoside” means a nucleoside comprising a2′-H(H) deoxyribosyl sugar moiety. In certain embodiments, a2′-deoxyribonucleoside is a 2′-β-D deoxyribonucleoside and comprises a2′β-D-deoxyribosyl sugar moiety, which has the β-D configuration asfound in naturally occurring deoxyribonucleic acids (DNA). In certainembodiments, a 2′-deoxyribonucleoside may comprise a modified nucleobaseor may comprise an RNA nucleobase (uracil).

As used herein, “2′-MOE” means a 2′-OCH₂CH₂OCH₃ group in place of the2′-OH group of a ribosyl sugar moiety. A “2′-MOE sugar moiety” is asugar moiety with a 2′-OCH₂CH₂OCH₃ group in place of the 2′-OH group ofa ribosyl sugar moiety. Unless otherwise indicated, a 2′-MOE sugarmoiety is in the β-D configuration. “MOE” means 0-methoxyethyl.

As used herein, “2′-MOE nucleoside” means a nucleoside comprising a2′-MOE sugar moiety.

As used herein, “2′-NMA” means a —O—CH₂—C(═O)—NH—CH₃ group in place ofthe 2′-OH group of a ribosyl sugar moiety. A “2′-NMA sugar moiety” is asugar moiety with a 2′-O—CH₂—C(═O)—NH—CH₃ group in place of the 2′-OHgroup of a ribosyl sugar moiety. Unless otherwise indicated, a 2′-NMAsugar moiety is in the β-D configuration. “NMA” means 0-N-methylacetamide.

As used herein, “2′-NMA nucleoside” means a nucleoside comprising a2′-NMA sugar moiety.

As used herein, “2′-OMe” means a 2′-OCH₃ group in place of the 2′-OHgroup of a ribosyl sugar moiety. A “2′-OMe sugar moiety” is a sugarmoiety with a 2′-OCH₃ group in place of the 2′-OH group of a ribosylsugar moiety. Unless otherwise indicated, a 2′-OMe sugar moiety is inthe β-D configuration. “OMe” means O-methyl.

As used herein, “2′-OMe nucleoside” means a nucleoside comprising a2′-OMe sugar moiety. As used herein, “2′-substituted nucleoside” means anucleoside comprising a 2′-substituted sugar moiety. As used herein,“2′-substituted” in reference to a sugar moiety means a sugar moietycomprising at least one 2′-substituent group other than H or OH.

As used herein, “5-methyl cytosine” means a cytosine modified with amethyl group attached to the 5 position. A 5-methyl cytosine is amodified nucleobase.

As used herein, “administering” means providing a pharmaceutical agentto a subject.

As used herein, “ameliorate” in reference to a treatment meansimprovement in at least one symptom relative to the same symptom in theabsence of the treatment. In certain embodiments, amelioration is thereduction in the severity or frequency of a symptom, or the delayedonset or slowing of progression in the severity or frequency of asymptom. In certain embodiments, the symptom is reduced muscle strength;inability or reduced ability to sit upright, to stand, and/or walk;reduced neuromuscular activity; reduced electrical activity in one ormore muscles; reduced respiration; inability or reduced ability to eat,drink, and/or breathe without assistance; loss of weight or reducedweight gain; and/or decreased survival.

As used herein, “antisense activity” means any detectable and/ormeasurable change attributable to the hybridization of an antisensecompound to its target nucleic acid.

As used herein, “antisense compound” means an oligomeric compound oroligomeric duplex capable of achieving at least one antisense activity.

As used herein, “bicyclic nucleoside” or “BNA” means a nucleosidecomprising a bicyclic sugar moiety.

As used herein, “bicyclic sugar” or “bicyclic sugar moiety” means amodified sugar moiety comprising two rings, wherein the second ring isformed via a bridge connecting two of the atoms in the first ringthereby forming a bicyclic structure. In certain embodiments, the firstring of the bicyclic sugar moiety is a furanosyl moiety. In certainembodiments, the furanosyl moiety is a ribosyl moiety. In certainembodiments, the bicyclic sugar moiety does not comprise a furanosylmoiety.

As used herein, “cerebrospinal fluid” or “CSF” means the fluid fillingthe space around the brain and spinal cord. “Artificial cerebrospinalfluid” or “aCSF” means a prepared or manufactured fluid that has certainproperties of cerebrospinal fluid.

As used herein, “cEt” means a 4′ to 2′ bridge in place of the 2′OH-groupof a ribosyl sugar moiety, wherein the bridge has the formula of4′-CH(CH₃)—O-2′, and wherein the methyl group of the bridge is in the Sconfiguration. A “cEt sugar moiety” is a bicyclic sugar moiety with a 4′to 2′ bridge in place of the 2′OH-group of a ribosyl sugar moiety,wherein the bridge has the formula of 4′-CH(CH₃)—O-2′, and wherein themethyl group of the bridge is in the S configuration. “cEt” meansconstrained ethyl.

As used herein, “cEt nucleoside” means a nucleoside comprising a cEtsugar moiety.

As used herein, “chirally enriched population” means a plurality ofmolecules of identical molecular formula, wherein the number orpercentage of molecules within the population that contain a particularstereochemical configuration at a particular chiral center is greaterthan the number or percentage of molecules expected to contain the sameparticular stereochemical configuration at the same particular chiralcenter within the population if the particular chiral center werestereorandom. Chirally enriched populations of molecules having multiplechiral centers within each molecule may contain one or more stereorandomchiral centers. In certain embodiments, the molecules are modifiedoligonucleotides. In certain embodiments, the molecules are compoundscomprising modified oligonucleotides.

As used herein, “complementary” in reference to an oligonucleotide meansthat at least 70% of the nucleobases of the oligonucleotide or one ormore portions thereof and the nucleobases of another nucleic acid or oneor more portions thereof are capable of hydrogen bonding with oneanother when the nucleobase sequence of the oligonucleotide and theother nucleic acid are aligned in opposing directions. Complementarynucleobases means nucleobases that are capable of forming hydrogen bondswith one another. Complementary nucleobase pairs include adenine (A)with thymine (T), adenine (A) with uracil (U), cytosine (C) with guanine(G), and 5-methyl cytosine (mC) with guanine (G). Complementaryoligonucleotides and/or target nucleic acids need not have nucleobasecomplementarity at each nucleoside. Rather, some mismatches aretolerated. As used herein, “fully complementary” or “100% complementary”in reference to an oligonucleotide, or a portion thereof, means that theoligonucleotide, or portion thereof, is complementary to anotheroligonucleotide or target nucleic acid at each nucleobase of the shorterof the two oligonucleotides, or at each nucleoside if theoligonucleotides are the same length.

As used herein, “contiguous” in the context of an oligonucleotide refersto nucleosides, nucleobases, sugar moieties, or internucleoside linkagesthat are immediately adjacent to each other. For example, “contiguousnucleobases” means nucleobases that are immediately adjacent to eachother in a sequence.

As used herein, “hybridization” means the pairing or annealing ofcomplementary oligonucleotides and/or nucleic acids. While not limitedto a particular mechanism, the most common mechanism of hybridizationinvolves hydrogen bonding, which may be Watson-Crick, Hoogsteen orreversed Hoogsteen hydrogen bonding, between complementary nucleobases.

As used herein, “internucleoside linkage” means the covalent linkagebetween contiguous nucleosides in an oligonucleotide. As used herein,“modified internucleoside linkage” means any internucleoside linkageother than a phosphodiester internucleoside linkage. “Phosphorothioateinternucleoside linkage” is a modified internucleoside linkage in whichone of the non-bridging oxygen atoms of a phosphodiester internucleosidelinkage is replaced with a sulfur atom.

As used herein, “mismatch” or “non-complementary” means a nucleobase ofa first oligonucleotide that is not complementary with the correspondingnucleobase of a second oligonucleotide or target nucleic acid when thefirst and second oligonucleotide are aligned.

As used herein, “motif” means the pattern of unmodified and/or modifiedsugar moieties, nucleobases, and/or internucleoside linkages, in anoligonucleotide.

As used herein, “non-bicyclic modified sugar moiety” means a modifiedsugar moiety that comprises a modification, such as a substituent, thatdoes not form a bridge between two atoms of the sugar to form a secondring.

As used herein, “nucleobase” means an unmodified nucleobase or amodified nucleobase. As used herein an “unmodified nucleobase” isadenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). Asused herein, a “modified nucleobase” is a group of atoms other thanunmodified A, T, C, U, or G capable of pairing with at least oneunmodified nucleobase. A “5-methyl cytosine” is a modified nucleobase. Auniversal base is a modified nucleobase that can pair with any one ofthe five unmodified nucleobases. As used herein, “nucleobase sequence”means the order of contiguous nucleobases in a target nucleic acid oroligonucleotide independent of any sugar or internucleoside linkagemodification.

As used herein, “nucleoside” means a compound comprising a nucleobaseand a sugar moiety. The nucleobase and sugar moiety are each,independently, unmodified or modified. As used herein, “modifiednucleoside” means a nucleoside comprising a modified nucleobase and/or amodified sugar moiety. “Linked nucleosides” are nucleosides that areconnected in a contiguous sequence (i.e., no additional nucleosides arepresented between those that are linked).

As used herein, “oligomeric compound” means an oligonucleotide andoptionally one or more additional features, such as a conjugate group orterminal group. An oligomeric compound may be paired with a secondoligomeric compound that is complementary to the first oligomericcompound or may be unpaired. A “singled-stranded oligomeric compound” isan unpaired oligomeric compound. The term “oligomeric duplex” means aduplex formed by two oligomeric compounds having complementarynucleobase sequences. Each oligomeric compound of an oligomeric duplexmay be referred to as a “duplexed oligomeric compound.”

As used herein, “oligonucleotide” means a strand of linked nucleosidesconnected via internucleoside linkages, wherein each nucleoside andinternucleoside linkage may be modified or unmodified. Unless otherwiseindicated, oligonucleotides consist of 8-50 linked nucleosides. As usedherein, “modified oligonucleotide” means an oligonucleotide, wherein atleast one nucleoside or internucleoside linkage is modified. As usedherein, “unmodified oligonucleotide” means an oligonucleotide that doesnot comprise any nucleoside modifications or internucleosidemodifications.

As used herein, “pharmaceutical composition” means a mixture ofsubstances suitable for administering to a subject. For example, apharmaceutical composition may comprise an oligomeric compound and asterile aqueous solution.

As used herein, “pharmaceutically acceptable carrier or diluent” meansany substance suitable for use in administering to a subject. Certainsuch carriers enable pharmaceutical compositions to be formulated as,for example, tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspension, and lozenges for the oral ingestion by a subject.In certain embodiments, a pharmaceutically acceptable carrier or diluentis sterile water, sterile saline, sterile buffer solution, or sterileartificial cerebrospinal fluid.

As used herein, “pharmaceutically acceptable salts” meansphysiologically and pharmaceutically acceptable salts of compounds.Pharmaceutically acceptable salts retain the desired biological activityof the parent compound and do not impart undesired toxicological effectsthereto.

As used herein, “RNA” means an RNA transcript and includes pre-mRNA andmature mRNA unless otherwise specified.

As used herein, “stereorandom chiral center” in the context of apopulation of molecules of identical molecular formula means a chiralcenter having a random stereochemical configuration. For example, in apopulation of molecules comprising a stereorandom chiral center, thenumber of molecules having the (5) configuration of the stereorandomchiral center may be but is not necessarily the same as the number ofmolecules having the (R) configuration of the stereorandom chiralcenter. The stereochemical configuration of a chiral center isconsidered random when it is the result of a synthetic method that isnot designed to control the stereochemical configuration. In certainembodiments, a stereorandom chiral center is a stereorandomphosphorothioate internucleoside linkage.

As used herein, “subject” means a human or non-human animal.

As used herein, “sugar moiety” means an unmodified sugar moiety or amodified sugar moiety. As used herein, “unmodified sugar moiety” means a2′-OH(H) β-D ribosyl moiety, as found in RNA (an “unmodified RNA sugarmoiety”), or a 2′-H(H) β-D deoxyribosyl moiety, as found in DNA (an“unmodified DNA sugar moiety”). Unmodified sugar moieties have onehydrogen at each of the 1′, 3′, and 4′ positions, an oxygen at the 3′position, and two hydrogens at the 5′ position. As used herein,“modified sugar moiety” or “modified sugar” means a modified furanosylsugar moiety or a sugar surrogate.

As used herein, “sugar surrogate” means a modified sugar moiety havingother than a furanosyl moiety that can link a nucleobase to anothergroup, such as an internucleoside linkage, conjugate group, or terminalgroup in an oligonucleotide. Modified nucleosides comprising sugarsurrogates can be incorporated into one or more positions within anoligonucleotide and such oligonucleotides are capable of hybridizing tocomplementary oligomeric compounds or target nucleic acids.

As used herein, “standard in vivo assay” means the assay described inExample 2 and reasonable variations thereof.

As used herein, “symptom” means any physical feature or test result thatindicates the existence or extent of a disease or disorder. In certainembodiments, a symptom is apparent to a subject or to a medicalprofessional examining or testing the subject.

As used herein, “target nucleic acid” means a nucleic acid that anantisense compound is designed to affect.

As used herein, “target region” means a portion of a target nucleic acidto which an oligomeric compound is designed to hybridize.

As used herein, “terminal group” means a chemical group or group ofatoms that is covalently linked to a terminus of an oligonucleotide.

As used herein, “therapeutically effective amount” means an amount of apharmaceutical agent that provides a therapeutic benefit to a subject.For example, a therapeutically effective amount improves a symptom of adisease.

Certain Embodiments

The present disclosure provides the following non-limiting numberedembodiments:

Embodiment 1. An oligomeric compound comprising a modifiedoligonucleotide consisting of 16, 17, 18, 19, or 20 linked nucleosidesand having a nucleobase sequence comprising at least 15 or at least 16contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs:20-50, wherein the modified oligonucleotide comprises at least onemodification selected from a modified sugar moiety and a modifiedinternucleoside linkage.

Embodiment 2. An oligomeric compound comprising a modifiedoligonucleotide consisting of 17, 18, 19, or 20 linked nucleosides andhaving a nucleobase sequence comprising at least 15, at least 16, or atleast 17 contiguous nucleobases of any of the nucleobase sequences ofSEQ ID NOs: 20-27, 29-30, or 32-50, wherein the modified oligonucleotidecomprises at least one modification selected from a modified sugarmoiety and a modified internucleoside linkage.

Embodiment 3. An oligomeric compound comprising a modifiedoligonucleotide consisting of 18, 19, or 20 linked nucleosides andhaving a nucleobase sequence comprising at least 15, at least 16, or atleast 17, or at least 18 contiguous nucleobases of any of the nucleobasesequences of SEQ ID NOs: 20-27, 30, or 33-50, wherein the modifiedoligonucleotide comprises at least one modification selected from amodified sugar moiety and a modified internucleoside linkage.

Embodiment 4. An oligomeric compound comprising a modifiedoligonucleotide consisting of 19 or 20 linked nucleosides and having anucleobase sequence comprising at least 15, at least 16, or at least 17,at least 18, or at least 19 contiguous nucleobases of any of thenucleobase sequences of SEQ ID NOs: 20, 22, 24-27, 30, 33-50, whereinthe modified oligonucleotide comprises at least one modificationselected from a modified sugar moiety and a modified internucleosidelinkage.

Embodiment 5. An oligomeric compound comprising a modifiedoligonucleotide consisting of 20 linked nucleosides and having anucleobase sequence comprising at least 15, at least 16, or at least 17,at least 18, at least 19, or at least 20 contiguous nucleobases of anyof the nucleobase sequences of SEQ ID NOs: 20, 22, 25, 27, 35, 39-46, or49, wherein the modified oligonucleotide comprises at least onemodification selected from a modified sugar moiety and a modifiedinternucleoside linkage.

Embodiment 6. The oligomeric compound of any of embodiments 1-5, whereinthe modified oligonucleotide has a nucleobase sequence that is at least80%, 85%, 87.5%, 88.2%, 89%, 89.4%, 90%, 93.7%, 94%, 94.7%, 95%, or is100% complementary to the nucleobase sequence of SEQ ID NO: 1 whenmeasured across the entire nucleobase sequence of the modifiedoligonucleotide.

Embodiment 7. The oligomeric compound of any of embodiments 1-6, whereinthe modified oligonucleotide has an internucleoside linkage motif (5′ to3′) selected from: sososssssssssssss, ssosssssssssssoss,ssosssssosssssoss, ssosssosssosssoss, soossssssssssooss,sooosssssssssooss, sooossssssssoooss, sssssssooosssssss,ssossssssssssssss, ssssossssssssssss, ssssssossssssssss,ssssssssossssssss, ssssssssssossssss, ssssssssssssossss,ssssssssssssssoss, sossssssssssssoss, sosssssssssosssss,sosssssssosssssss, sosssssosssssssss, sosssosssssssssss,ssssosssssssssoss, ssssssosssssssoss, ssssssssosssssoss,ssssssssssosssoss, ssssssssssssososs, soossssssssssssss,sssoossssssssssss, sssssoossssssssss, sssssssoossssssss,sssssssssoossssss, sssssssssssoossss, sssssssssssssooss,sssssssoooossssss, ssoooosssssssssss, ssssoooosssssssss,ssssssssoooosssss, ssssssssssoooosss, sssssssssssooooss,ssssssooooossssss, ssssssoooooosssss, soooosssssssoooss,sssssooooooosssss, ssssssssssssssoss, ssssssssssssosss,sssssssssssssooss, ssssssssssssososs, sssssssssssosssss,sssssssssssososss, ssssssssssossosss, sssssssssosssssss,sssssssssosssosss, ssssssssosssssoss, ssssssssossssosss,sssssssosssssssss, sssssssoossssssss, SSSSSOSSSSSSSSSSS,sssosssssssssssss, sosssssssssssssss, sossssssossssssss,soossssssssssssss, ossssssssssssssssso, sssssssssssssssssoo,sssssssssossssssoss, sssssssssssssssooss, ssssssssssssssososs,sssssssssosssssssss, sssssssssossssssoss, ssssssssoosssssssss,sosssssssssssssssss, sossssssssssssssoss, sosssssssosssssssss,sososssssssssssssss, soossssssssssssssss, sssssssssssssssssss,ssssssssssssssssso, ossssssssssssssssss, ssssssssssssososso,sssssssssssssssoss, sssssssssssssososs, ssssssssosssssssss,ssssssssossssssoss, sossssssssssssssss, sosssssssssssssoss,sossssssosssssssss, sosossssssssssssss, ssssssssssooooss,ssssssssoooossss, sssssssooossssss, ssssssoooossssss, ssssssooooosssss,sssssoooooosssss, sssssooooooossss, ssssoooossssssss, ssossssssssssoss,ssosssssossssoss, ssosssosssossoss, ssossossossososs, ssososososososss,ssoooossssssssss, soosssssssssooss, sooossssssssooss, sooosssssssoooss,soooossssssoooss, sssssssssooooss, ssssssssoooosss, ssssssooossssss,ssssssoooosssss, sssssooooosssss, sssssoooooossss, ssssoooosssssss,ssssooooooossss, sssosssosssosss, ssosssssssssoss, ssossossossosss,ssossossosososs, ssososososososs, ssoooosssssssss, soossssssssooss,sooosssssssooss, sooossssssoooss, and soooosssssoooss; wherein ‘s’represents a phosphorothioate internucleoside linkage and ‘o’ representsa phosphodiester internucleoside linkage.

Embodiment 8. The oligomeric compound of any of embodiments 1-6, whereinthe modified oligonucleotide has an internucleoside linkage motifselected from: sssssssssssssssxs and ssssssssssssssssx, wherein ‘s’represents a phosphorothioate internucleoside linkage, ‘o’ represents aphosphodiester internucleoside linkage, and “x” represents amethoxypropylphosphonate internucleoside linkage.

Embodiment 9. The oligomeric compound of any of embodiments 1-6, whereinthe modified oligonucleotide has an internucleoside linkage motifselected from zzzzzzzzzzzzzzzzz, ssssssssssszzzzzz, ssssszzzzzzssssss,zzooooooooooooozz, zzzzooooooooooozz, zzzzzzooooooooozz,zzzzzzzzooooooozz, and ssoooooooooooooss, wherein ‘s’ represents aphosphorothioate internucleoside linkage, ‘o’ represents aphosphodiester internucleoside linkage, and “z” represents a mesylphosphoramidate internucleoside linkage.

Embodiment 10. The oligomeric compound of any of embodiments 1-9,wherein the modified oligonucleotide has a sugar motif (5′ to 3′)selected from: eeeeeeeeeeeeeeeeeeee, eeeeeeeeeeeeeeeeeee,eeeeeeeeeeeeeeeeee, eeeeeeeeeeeeeeeee, eeeeeeeeeeeeeeee,nnnnnnnnnnnnnnnn, nnnnnnnnnnnnnnnnn, nnnnnnnnnnnnnnnnnn,nnnnnnnnnnnnnnnnnnn, nnnnnnnnnnnnnnnnnnnn, nennnnneneennnnnnn,nnnnnnnnnnnnenneen, nennnnneneenenneen, nnnnnnnnnnnnnnnnnnne,nnnnnnnnnnnnnnnnnnnd, nnnnnnnnnnnnnnnnnnny, nnnnnnnnnnnnnnnndd,nnnnnnnnnnnnnnnned, nnnnnnnnnnnnnnnnde, nnnnnnnnnnnnnnnnee,eeeeeeeeeeeeeeeeeed, keekeekeekeekeeeek, keeekeeekeeekeeeek,keeeeekeeeeekeeeek, keeeeeeekeeeeeeeek, keeeeeeeeeeeeeeeek,eeekeekeekeekeekek, eeekeekeekeekeekee, eeeeeekeekeekeekee,eeeeeekeekeekeeeee, eeeeeekeeeeekeeeee, keekeekeekeeeeeeee,eeeeeeeekeekeekeek, keekeekeeeeeeeeeee, eeeeeeeeeeekeekeek,keekeeeeeeeeeeeeee, eeeeeeeeeeeeeekeek, keekeekeekeekeeek,keeekeeekeeekeeek, keeeekeeeeekeeeek, keeeeeeekeeeeeeek,keeeeeeeeeeeeeeek, eekeekeekeekeekek, eekeekeekeekeekee,eeeeekeekeekeekee, eeeeekeekeekeeeee, eeeeekeeeeekeeeee,keekeekeekeeeeeee, eeeeeeekeekeekeek, keekeekeeeeeeeeee,eeeeeeeeeekeekeek, keekeeeeeeeeeeeee, eeeeeeeeeeeeekeek,keekeekeekeekeek, keeekeeekeeekeek, keeeekeeeekeeeek, keeeeeeekeeeeeek,keeeeeeeeeeeeeek, kekeekeekeekeeke, eekeekeekeekeeke, eeeeekeekeekeeke,eeeeekeekeekeeee, eeeeekeeeeekeeee, keekeekeekeeeeee, eeeeeekeekeekeek,keekeekeeeeeeeee, eeeeeeeeekeekeek, keekeeeeeeeeeeee, eeeeeeeeeeeekeek,eeeeeeeeeeeeeeeeeed, eeeeeeeeeeeeeeeeeey, ennnnnnnnnnnnnnnnnn, andennnnnnnnnnnnnnnnnne; wherein ‘e’ represents a 2′-MOE sugar moiety, ‘n’represents a 2′-NMA sugar moiety, ‘k’ represents a cEt sugar moiety, ‘d’represents a 2′β-D-deoxyribosyl sugar moiety, and ‘y’ represents a2′-OMe sugar moiety.

Embodiment 11. The oligomeric compound of any of embodiments 1-9,wherein the modified oligonucleotide has a sugar motif (5′ to 3′)selected from nnnnnnnnnnnnnnnenn and nnnnnnnnnnnnnnnnen, wherein ‘e’represents a 2′-MOE sugar moiety and ‘n’ represents a 2′-NMA sugarmoiety.

Embodiment 12. The oligomeric compound of any of embodiments 1-9,wherein the modified oligonucleotide has a sugar motif (5′ to 3′) ofqqnqqqqqnqnnqnqqnn, wherein in each ‘n’ represents a 2′-NMA sugarmoiety, and each ‘q’ is independently selected from a2′-O—(N,N-dimethyl) acetamide sugar moiety, a 2′-O—(N-ethyl) acetamidesugar moiety, a 2′-O—(N-propyl) acetamide sugar moiety, a2′O—(N-cyclopropyl) acetamide sugar moiety, and a2′-O—(N-cyclopropylmethyl) acetamide sugar moiety.

Embodiment 13. The oligomeric compound of any of embodiments 1-9,wherein the modified oligonucleotide comprises at least one modifiedsugar moiety.

Embodiment 14. The oligomeric compound of embodiment 13, wherein themodified oligonucleotide comprises at least one bicyclic sugar moiety.

Embodiment 15. The oligomeric compound of embodiment 14, wherein thebicyclic sugar moiety has a 4′-2′ bridge, wherein the 4′-2′ bridge isselected from —CH₂—O—; and —CH(CH₃)—O—.

Embodiment 16. The oligomeric compound of embodiment 13, wherein themodified oligonucleotide comprises at least one non-bicyclic modifiedsugar moiety.

Embodiment 17. The oligomeric compound of embodiment 16, wherein thenon-bicyclic modified sugar moiety is any of a 2′-MOE sugar moiety, a2′-NMA sugar moiety, a 2′-OMe sugar moiety, or a 2′-F sugar moiety.

Embodiment 18. The oligomeric compound of embodiment 13, wherein themodified oligonucleotide comprises at least one sugar surrogate.

Embodiment 19. The oligomeric compound of embodiment 18, wherein thesugar surrogate is any of morpholino, modified morpholino, PNA, THP, andF-HNA.

Embodiment 20. The oligomeric compound of any of embodiments 1-6 and10-19, wherein the modified oligonucleotide comprises at least onemodified internucleoside linkage.

Embodiment 21. The oligomeric compound of embodiment 20, wherein eachinternucleoside linkage of the modified oligonucleotide is a modifiedinternucleoside linkage.

Embodiment 22. The oligomeric compound of embodiment 20 or embodiment21, wherein the modified internucleoside linkage is a phosphorothioateinternucleoside linkage.

Embodiment 23. The oligomeric compound of any of embodiments 1-20 or 22,wherein the modified oligonucleotide comprises at least onephosphodiester internucleoside linkage.

Embodiment 24. The oligomeric compound of any of embodiments 20, 22, or23, wherein each internucleoside linkage is independently selected froma phosphodiester internucleoside linkage and a phosphorothioateinternucleoside linkage.

Embodiment 25. The oligomeric compound of any of embodiments 13-19,wherein the modified oligonucleotide has an internucleoside linkagemotif (5′ to 3′) selected from: sososssssssssssss, soossssssssssssss,sosssosssssssssss, sosssssosssssssss, sosssssssosssssss,sssoossssssssssss, sssssssoossssssss, sssssssssoossssss, andsssssssssssoossss; wherein ‘s’ represents a phosphorothioateinternucleoside linkage and ‘o’ represents a phosphodiesterinternucleoside linkage.

Embodiment 26. The oligomeric compound of any of embodiments 1-25,wherein the modified oligonucleotide comprises a modified nucleobase.

Embodiment 27. The oligomeric compound of embodiment 26, wherein themodified nucleobase is a 5-methyl cytosine.

Embodiment 28. The oligomeric compound of any of embodiments 1-27,wherein the modified oligonucleotide consists of 16, 17, 18, 19, or 20linked nucleosides.

Embodiment 29. The oligomeric compound of any of embodiments 1-28,wherein the modified oligonucleotide comprises 1 or 2 non-complementarynucleobases.

Embodiment 30. The oligomeric compound of any of embodiments 1-29,wherein the modified oligonucleotide comprises 1 or 2 cleavablemoieties.

Embodiment 31. The oligomeric compound of embodiment 30, wherein thecleavable moiety is a phophodiester internucleoside linkage.

Embodiment 32. The oligomeric compound of any of embodiments 1-31,consisting of the modified oligonucleotide.

Embodiment 33. The oligomeric compound of any of embodiments 1-32,wherein the oligomeric compound is a singled-stranded oligomericcompound.

Embodiment 34. An oligomeric compound comprising a modifiedoligonucleotide according to the following chemical notation: ^(m)C_(es)A_(eo) ^(m)C_(es) T_(eo) T_(es) T_(es) ^(m)C_(es) A_(es) T_(es) A_(es)A_(es) T_(es) G_(es) ^(m)C_(es) T_(es) G_(es) G_(es) ^(m)C_(e) (SEQ IDNO: 21), wherein:

A=an adenine nucleobase,

^(m)C=a 5-methyl cytosine nucleobase,

G=a guanine nucleobase,

T=a thymine nucleobase,

e=a 2′-MOE sugar moiety,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

Embodiment 35. An oligomeric compound comprising a modifiedoligonucleotide according to the following chemical notation: T_(eo)T_(es) ^(m)C_(es) A_(es) ^(m)C_(es) T_(es) T_(es) T_(es) ^(m)C_(es)A_(es) T_(es) A_(es) A_(es) T_(es) G_(es) ^(m)C_(es) T_(es) G_(es)G_(eo) ^(m)C_(e) (SEQ ID NO: 22), wherein:

A=an adenine nucleobase,

m_(C)=a 5-methyl cytosine nucleobase,

G=a guanine nucleobase,

T=a thymine nucleobase,

e=a 2′-MOE sugar moiety,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

Embodiment 36. An oligomeric compound comprising a modifiedoligonucleotide according to the following chemical notation: T_(eo)T_(os) ^(m)C_(ns) A_(os) ^(m)C_(ns) T_(os) T_(os) T_(os) ^(m)C_(ns)A_(os) T_(os) A_(os) A_(os) T_(os) G_(os) ^(m)C_(ns) T_(os) G_(os)G_(no) ^(m)C_(e) (SEQ ID NO: 22), wherein:

A=an adenine nucleobase,

^(m)C=_(a) 5-methylcytosine nucleobase,

G=a guanine nucleobase,

T=a thymine nucleobase,

e=a 2′-MOE sugar moiety,

n=a 2′-NMA sugar moiety,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

Embodiment 37. An oligomeric compound comprising a modifiedoligonucleotide according to the following chemical notation: ^(m)C_(ns)A_(no) ^(m)C_(ns) T_(no) T_(ns) T_(ns) ^(m)C_(ns) A_(ns) T_(ns) A_(ns)A_(ns) T_(ns) G_(ns) ^(m)C_(ns) T_(ns) G_(ns) G_(ns) ^(m)C_(n) (SEQ IDNO: 21), wherein:

A=an adenine nucleobase,

^(m)C=a 5-methyl cytosine nucleobase,

G=a guanine nucleobase,

T=a thymine nucleobase,

n=a 2′-NMA sugar moiety,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

Embodiment 38. A modified oligonucleotide according to the followingchemical structure:

or a salt thereof.

Embodiment 39. The modified oligonucleotide of embodiment 38, which isthe sodium salt or the potassium salt.

Embodiment 40. A modified oligonucleotide according to the followingchemical structure:

Embodiment 41. A modified oligonucleotide according to the followingchemical structure:

or a salt thereof.

Embodiment 42. The modified oligonucleotide of embodiment 41, which isthe sodium salt or the potassium salt.

Embodiment 43. A modified oligonucleotide according to the followingchemical structure:

Embodiment 44. A modified oligonucleotide according to the followingchemical structure:

or a salt thereof.

Embodiment 45. The modified oligonucleotide of embodiment 44, which isthe sodium salt or the potassium salt.

Embodiment 46. A modified oligonucleotide corresponding to the followingchemical structure:

Embodiment 47. A modified oligonucleotide according to the followingchemical structure:

or a salt thereof.

Embodiment 48. The modified oligonucleotide of embodiment 47, which isthe sodium salt or the potassium salt.

Embodiment 49. A modified oligonucleotide according to the followingchemical structure:

Embodiment 50. A pharmaceutical composition comprising the oligomericcompound of any of embodiments 1-36 or the modified oligonucleotide ofany of embodiments 38-49, and a pharmaceutically acceptable diluent orcarrier.

Embodiment 51. The pharmaceutical composition of embodiment 50,comprising a pharmaceutically acceptable diluent and wherein thepharmaceutically acceptable diluent is artificial CSF (aCSF) or PBS.

Embodiment 52. The pharmaceutical composition of embodiment 51, whereinthe pharmaceutical composition consists essentially of the modifiedoligonucleotide and artificial CSF (aCSF).

Embodiment 53. The pharmaceutical composition of embodiment 51, whereinthe pharmaceutical composition consists essentially of the modifiedoligonucleotide and PBS.

Embodiment 54. A chirally enriched population of modifiedoligonucleotides of any of embodiments 38-49, wherein the population isenriched for modified oligonucleotides comprising at least oneparticular phosphorothioate internucleoside linkage having a particularstereochemical configuration.

Embodiment 55. The chirally enriched population of embodiment 54,wherein the population is enriched for modified oligonucleotidescomprising at least one particular phosphorothioate internucleosidelinkage having the (Sp) configuration.

Embodiment 56. The chirally enriched population of embodiment 54,wherein the population is enriched for modified oligonucleotidescomprising at least one particular phosphorothioate internucleosidelinkage having the (Rp) configuration.

Embodiment 57. The chirally enriched population of embodiment 54,wherein the population is enriched for modified oligonucleotides havinga particular, independently selected stereochemical configuration ateach phosphorothioate internucleoside linkage.

Embodiment 58. The chirally enriched population of embodiment 57,wherein the population is enriched for modified oligonucleotides havingthe (Sp) configuration at each phosphorothioate internucleoside linkageor for modified oligonucleotides having the (Rp) configuration at eachphosphorothioate internucleoside linkage.

Embodiment 59. The chirally enriched population of embodiment 57,wherein the population is enriched for modified oligonucleotides havingthe (Rp) configuration at one particular phosphorothioateinternucleoside linkage and the (Sp) configuration at each of theremaining phosphorothioate internucleoside linkages.

Embodiment 60. The chirally enriched population of embodiment 57,wherein the population is enriched for modified oligonucleotides havingat least 3 contiguous phosphorothioate internucleoside linkages in theSp, Sp, and Rp configurations, in the 5′ to 3′ direction.

Embodiment 61. A population of modified oligonucleotides of any ofembodiments 38-49, wherein all of the phosphorothioate internucleosidelinkages of the modified oligonucleotide are stereorandom.

Embodiment 62. A method of treating a disease associated with SMN1 orSMN2 comprising administering to a subject having or at risk fordeveloping a disease associated with SMN1 or SMN2 a therapeuticallyeffective amount of a pharmaceutical composition according to any ofembodiments 50-53; and thereby treating the disease associated with SMN1or SMN2.

Embodiment 63. The method of embodiment 62, wherein the diseaseassociated with SMN1 or SMN2 is a neurodegenerative disease.

Embodiment 64. The method of embodiment 63, wherein theneurodegenerative disease is Spinal Muscular Atrophy (SMA).

Embodiment 65. The method of embodiment 64, wherein the SMA is any ofType I SMA, Type II SMA, Type III SMA, or Type IV SMA.

Embodiment 66. The method of embodiment 64 or embodiment 65, wherein atleast one symptom of SMA is ameliorated.

Embodiment 67. The method of embodiment 66, wherein the symptom is anyof reduced muscle strength; inability or reduced ability to sit upright,to stand, and/or walk; reduced neuromuscular activity; reducedelectrical activity in one or more muscles; reduced respiration;inability or reduced ability to eat, drink, and/or breathe withoutassistance; loss of weight or reduced weight gain; and/or decreasedsurvival.

Embodiment 68. The method of any of embodiments 62-67, wherein thepharmaceutical composition is administered to the central nervous systemor systemically.

Embodiment 69. The method of embodiment 68, wherein the pharmaceuticalcomposition is administered to the central nervous system andsystemically.

Embodiment 70. The method of any of embodiment 62-67, wherein thepharmaceutical composition is administered any of intrathecally,systemically, subcutaneously, or intramuscularly.

Embodiment 71. A method of increasing SMN2 RNA including exon 7comprising contacting a cell, tissue, or organ with an oligomericcompound of any of embodiments 1-37, a modified oligonucleotide of anyof embodiments 38-49, or a pharmaceutical composition of any ofembodiments 50-53.

Certain Oligonucleotides

In certain embodiments, provided herein are oligomeric compoundscomprising oligonucleotides, which consist of linked nucleosides.Oligonucleotides may be unmodified oligonucleotides (RNA or DNA) or maybe modified oligonucleotides. Modified oligonucleotides comprise atleast one modification relative to unmodified RNA or DNA. That is,modified oligonucleotides comprise at least one modified nucleoside(comprising a modified sugar moiety and/or a modified nucleobase) and/orat least one modified internucleoside linkage.

Certain Modified Nucleosides

Modified nucleosides comprise a modified sugar moiety or a modifiednucleobase or both a modified sugar moiety and a modified nucleobase.

Certain Sugar Moieties

In certain embodiments, modified sugar moieties are non-bicyclicmodified sugar moieties. In certain embodiments, modified sugar moietiesare bicyclic or tricyclic sugar moieties. In certain embodiments,modified sugar moieties are sugar surrogates. Such sugar surrogates maycomprise one or more substitutions corresponding to those of other typesof modified sugar moieties.

In certain embodiments, modified sugar moieties are non-bicyclicmodified sugar moieties comprising a furanosyl ring with one or moresubstituent groups none of which bridges two atoms of the furanosyl ringto form a bicyclic structure. Such non bridging substituents may be atany position of the furanosyl, including but not limited to substituentsat the 2′, 4′, and/or 5′ positions. In certain embodiments one or morenon-bridging substituent of non-bicyclic modified sugar moieties isbranched. Examples of 2′-substituent groups suitable for non-bicyclicmodified sugar moieties include but are not limited to: 2′-F, 2′-OCH₃(“OMe” or “O-methyl”), and 2′-O(CH₂)₂OCH₃ (“MOE” or “O-methoxyethyl”),and 2′-O—N-alkyl acetamide, e.g., 2′-O—N-methyl acetamide (“NMA”),2′-O—N-dimethyl acetamide, acetamide, or 2′-O—N-propyl acetamide. Forexample, see U.S. Pat. No. 6,147,200, Prakash et al., 2003, Org. Lett.,5, 403-6. A “2′-O—N-methyl acetamide nucleoside” or “2′-NMA nucleoside”is shown below:

In certain embodiments, 2′-substituent groups are selected from among:halo, allyl, amino, azido, SH, CN, OCN, CF₃, OCF₃, O—C₁-C₁₀ alkoxy,O—C₁-C₁₀ substituted alkoxy, O—C₁-C₁₀ alkyl, O—C₁-C₁₀ substituted alkyl,S-alkyl, N(R_(m))-alkyl, O-alkenyl, S-alkenyl, N(R_(m))-alkenyl,O-alkynyl, S-alkynyl, N(R_(m))-alkynyl, O-alkylenyl-O-alkyl, alkynyl,alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃,O(CH₂)₂ON(R_(m))(R_(n)) or OCH₂C(═O)—N(R_(m))(R_(n)), where each R_(m)and R_(n) is, independently, H, an amino protecting group, orsubstituted or unsubstituted C₁-C₁₀ alkyl, and the 2′-substituent groupsdescribed in Cook et al., U.S. Pat. No. 6,531,584; Cook et al., U.S.Pat. No. 5,859,221; and Cook et al., U.S. Pat. No. 6,005,087. Certainembodiments of these 2′-substituent groups can be further substitutedwith one or more substituent groups independently selected from among:hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂), thiol,thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl.Examples of 4′-substituent groups suitable for non-bicyclic modifiedsugar moieties include but are not limited to alkoxy (e.g., methoxy),alkyl, and those described in Manoharan et al., WO 2015/106128. Examplesof 5′-substituent groups suitable for non-bicyclic modified sugarmoieties include but are not limited to: 5′-methyl (R or S), 5′-vinyl,and 5′-methoxy. In certain embodiments, non-bicyclic modified sugarmoieties comprise more than one non-bridging sugar substituent, forexample, 2′-F-5′-methyl sugar moieties and the modified sugar moietiesand modified nucleosides described in Migawa et al., WO 2008/101157 andRajeev et al., US2013/0203836.

In certain embodiments, a 2′-substituted non-bicyclic modifiednucleoside comprises a sugar moiety comprising a non-bridging2′-substituent group selected from: F, NH₂, N₃, OCF₃, OCH₃, O(CH₂)₃NH₂,CH₂CH═CH₂, OCH₂CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O(CH₂)₂ON(R_(m))(R_(n)), O(CH₂), ON(CH₃)₂, O(CH₂)₂O(CH₂)₂N(CH₃)₂, andN-substituted acetamide (OCH₂C(═O)—N(R_(m))(R_(n))), where each R_(m)and R_(n) is, independently, H, an amino protecting group, orsubstituted or unsubstituted C₁-C₁₀ alkyl, e.g., for example,OCH₂C(═O)—N(H)CH₃ (“NMA”).

In certain embodiments, a 2′-substituted non-bicyclic modifiednucleoside comprises a sugar moiety comprising a non-bridging2′-substituent group selected from: F, OCF₃, OCH₃, OCH₂CH₂OCH₃,O(CH₂)₂SCH₃, O(CH₂)₂ON(CH₃)₂, O(CH₂)₂O(CH₂)₂N(CH₃)₂, andOCH₂C(═O)—N(H)CH₃ (“NMA”).

In certain embodiments, a 2′-substituted non-bicyclic modifiednucleoside comprises a sugar moiety comprising a non-bridging2′-substituent group selected from: F, OCH₃, OCH₂CH₂OCH₃, andOCH₂C(═O)—N(H)CH₃.

Certain modified sugar moieties comprise a substituent that bridges twoatoms of the furanosyl ring to form a second ring, resulting in abicyclic sugar moiety. In certain such embodiments, the bicyclic sugarmoiety comprises a bridge between the 4′ and the 2′ furanose ring atoms.Examples of such 4′ to 2′ bridging sugar substituents include but arenot limited to: 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′(“LNA”), 4′-CH₂—S-2′, 4′-(CH₂)₂—O-2′ (“ENA”), 4′-CH(CH₃)—O-2′ (referredto as “constrained ethyl” or “cEt”), 4′-CH₂—O—CH₂-2′, 4′-CH₂—N(R)-2′,4′-CH(CH₂OCH₃)—O-2′ (“constrained MOE” or “cMOE”) and analogs thereof(see, e.g., Seth et al., U.S. Pat. No. 7,399,845, Bhat et al., U.S. Pat.No. 7,569,686, Swayze et al., U.S. Pat. No. 7,741,457, and Swayze etal., U.S. Pat. No. 8,022,193), 4′-C(CH₃)(CH₃)—O-2′ and analogs thereof(see, e.g., Seth et al., U.S. Pat. No. 8,278,283), 4′-CH₂—N(OCH₃)-2′ andanalogs thereof (see, e.g., Prakash et al., U.S. Pat. No. 8,278,425),4′-CH₂—O—N(CH₃)-2′ (see, e.g., Allerson et al., U.S. Pat. No. 7,696,345and Allerson et al., U.S. Pat. No. 8,124,745), 4′-CH₂—C(H)(CH₃)-2′ (see,e.g., Zhou, et al., J. Org. Chem., 2009, 74, 118-134), 4′-CH₂—C(═CH₂)-2′and analogs thereof (see e.g., Seth et al., U.S. Pat. No. 8,278,426),4′-C(R_(a)R_(b))—N(R)—O-2′, 4′-C(R_(a)R_(b))—O—N(R)-2′,4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O- 2′, wherein each R, R_(a), andR_(b) is, independently, H, a protecting group, or C₁-C₁₂ alkyl (see,e.g. Imanishi et al., U.S. Pat. No. 7,427,672).

In certain embodiments, such 4′ to 2′ bridges independently comprisefrom 1 to 4 linked groups independently selected from:—[C(R_(a))(R_(b))]_(a)—, —[C(R_(a))(R_(b))]_(a)—O—, —C(R_(a))═C(R_(b))—,—C(R_(a))═N—, —C(═NR_(a))—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and—N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl,or a protecting group.

Additional bicyclic sugar moieties are known in the art, see, forexample: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443,Albaek et al., J. Org. Chem., 2006, 71, 7731-7740, Singh et al., Chem.Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54,3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222;Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al.,J. Am. Chem. Soc., 2007, 129, 8362-8379; Wengel et al., U.S. Pat. No.7,053,207; Imanishi et al., U.S. Pat. No. 6,268,490; Imanishi et al.U.S. Pat. No. 6,770,748; Imanishi et al., U.S. RE44,779; Wengel et al.,U.S. Pat. No. 6,794,499; Wengel et al., U.S. Pat. No. 6,670,461; Wengelet al., U.S. Pat. No. 7,034,133; Wengel et al., U.S. Pat. No. 8,080,644;Wengel et al., U.S. Pat. No. 8,034,909; Wengel et al., U.S. Pat. No.8,153,365; Wengel et al., U.S. Pat. No. 7,572,582; and Ramasamy et al.,U.S. Pat. No. 6,525,191; Torsten et al., WO 2004/106356; Wengel et al.,WO 1999/014226; Seth et al., WO 2007/134181; Seth et al., U.S. Pat. No.7,547,684; Seth et al., U.S. Pat. No. 7,666,854; Seth et al., U.S. Pat.No. 8,088,746; Seth et al., U.S. Pat. No. 7,750,131; Seth et al., U.S.Pat. No. 8,030,467; Seth et al., U.S. Pat. No. 8,268,980; Seth et al.,U.S. Pat. No. 8,546,556; Seth et al., U.S. Pat. No. 8,530,640; Migawa etal., U.S. Pat. No. 9,012,421; Seth et al., U.S. Pat. No. 8,501,805; andU.S. Patent Publication Nos. Allerson et al., US2008/0039618 and Migawaet al., US2015/0191727.

In certain embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, an LNA nucleoside (describedherein) may be in the α-L configuration or in the β-D configuration.

α-L-methyleneoxy (4′-CH₂—O-2′) or α-L-LNA bicyclic nucleosides have beenincorporated into oligonucleotides that showed antisense activity(Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). Herein,general descriptions of bicyclic nucleosides include both isomericconfigurations. When the positions of specific bicyclic nucleosides(e.g., LNA or cEt) are identified in exemplified embodiments herein,they are in the β-D configuration, unless otherwise specified.

In certain embodiments, modified sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars).

In certain embodiments, modified sugar moieties are sugar surrogates. Incertain such embodiments, the oxygen atom of the sugar moiety isreplaced, e.g., with a sulfur, carbon or nitrogen atom. In certain suchembodiments, such modified sugar moieties also comprise bridging and/ornon-bridging substituents as described herein. For example, certainsugar surrogates comprise a 4′-sulfur atom and a substitution at the2′-position (see, e.g., Bhat et al., U.S. Pat. No. 7,875,733 and Bhat etal., U.S. Pat. No. 7,939,677) and/or the 5′ position.

In certain embodiments, sugar surrogates comprise rings having otherthan 5 atoms. For example, in certain embodiments, a sugar surrogatecomprises a six-membered tetrahydropyran (“THP”). Such tetrahydropyransmay be further modified or substituted. Nucleosides comprising suchmodified tetrahydropyrans include but are not limited to hexitol nucleicacid (“HNA”), anitol nucleic acid (“ANA”), manitol nucleic acid (“MNA”)(see, e.g., Leumann, C J. Bioorg. & Med. Chem. 2002, 10, 841-854),fluoro HNA:

(“F-HNA”, see e.g. Swayze et al., U.S. Pat. No. 8,088,904; Swayze etal., U.S. Pat. No. 8,440,803; Swayze et al., U.S. Pat. No. 8,796,437;and Swayze et al., U.S. Pat. No. 9,005,906; F-HNA can also be referredto as a F-THP or 3′-fluoro tetrahydropyran), and nucleosides comprisingadditional modified THP compounds having the formula:

wherein, independently, for each of said modified THP nucleoside:

Bx is a nucleobase moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the modified THP nucleoside to the remainder of anoligonucleotide or one of T₃ and T₄ is an internucleoside linking grouplinking the modified THP nucleoside to the remainder of anoligonucleotide and the other of T₃ and T₄ is H, a hydroxyl protectinggroup, a linked conjugate group, or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, or substituted C₂-C₆ alkynyl; and

each of R₁ and R₂ is independently selected from among: hydrogen,halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₃, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁, and eachJ₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, modified THP nucleosides are provided whereinq₁, q₂, q3, q4, q5, q6 and q₇ are each H.

In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ isother than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄,q₅, q₆ and q₇ is methyl. In certain embodiments, modified THPnucleosides are provided wherein one of R₁ and R₂ is F. In certainembodiments, R₁ is F and R₂ is H, in certain embodiments, R₁ is methoxyand R₂ is H, and in certain embodiments, R₁ is methoxyethoxy and R₂ isH.

In certain embodiments, sugar surrogates comprise rings having more than5 atoms and more than one heteroatom. For example, nucleosidescomprising morpholino sugar moieties and their use in oligonucleotideshave been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41,4503-4510 and Summerton et al., U.S. Pat. No. 5,698,685; Summerton etal., U.S. Pat. No. 5,166,315; Summerton et al., U.S. Pat. No. 5,185,444;and Summerton et al., U.S. Pat. No. 5,034,506). As used here, the term“morpholino” means a sugar surrogate having the following structure:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.”

In certain embodiments, sugar surrogates comprise acyclic moieties.Examples of nucleosides and oligonucleotides comprising such acyclicsugar surrogates include but are not limited to: peptide nucleic acid(“PNA”), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org.Biomol. Chem., 2013, 11, 5853-5865), and nucleosides andoligonucleotides described in Manoharan et al., WO2011/133876.

Many other bicyclic and tricyclic sugar and sugar surrogate ring systemsare known in the art that can be used in modified nucleosides.

Certain Modified Nucleobases

In certain embodiments, modified oligonucleotides comprise one or morenucleosides comprising an unmodified nucleobase. In certain embodiments,modified oligonucleotides comprise one or more nucleosides comprising amodified nucleobase. In certain embodiments, modified oligonucleotidescomprise one or more nucleosides that does not comprise a nucleobase,referred to as an abasic nucleoside.

In certain embodiments, modified nucleobases are selected from:5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynylsubstituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6substituted purines. In certain embodiments, modified nucleobases areselected from: 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine,2-propyl adenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-propynyl (—C□C—CH₃) uracil, 5-propynylcytosine, 6-azouracil,6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-azaand other 8-substituted purines, 5-halo, particularly 5-bromo,5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine,7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine,7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine,2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases. Further modified nucleobases include tricyclicpyrimidines, such as 1,3-diazaphenoxazine-2-one,1,3-diazaphenothiazine-2-one and9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp) Modifiednucleobases may also include those in which the purine or pyrimidinebase is replaced with other heterocycles, for example 7-deazaadenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in Merigan et al., U.S. Pat. No. 3,687,808,those disclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859;Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications,Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and thosedisclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T.,Ed., CRC Press, 2008, 163-166 and 442-443.

Publications that teach the preparation of certain of the above notedmodified nucleobases as well as other modified nucleobases includewithout limitation, Manoharan et al., US2003/0158403; Manoharan et al.,US2003/0175906; Dinh et al., U.S. Pat. No. 4,845,205; Spielvogel et al.,U.S. Pat. No. 5,130,302; Rogers et al., U.S. Pat. No. 5,134,066;Bischofberger et al., U.S. Pat. No. 5,175,273; Urdea et al., U.S. Pat.No. 5,367,066; Benner et al., U.S. Pat. No. 5,432,272; Matteucci et al.,U.S. Pat. No. 5,434,257; Gmeiner et al., U.S. Pat. No. 5,457,187; Cooket al., U.S. Pat. No. 5,459,255; Froehler et al., U.S. Pat. No.5,484,908; Matteucci et al., U.S. Pat. No. 5,502,177; Hawkins et al.,U.S. Pat. No. 5,525,711; Haralambidis et al., U.S. Pat. No. 5,552,540;Cook et al., U.S. Pat. No. 5,587,469; Froehler et al., U.S. Pat. No.5,594,121; Switzer et al., U.S. Pat. No. 5,596,091; Cook et al., U.S.Pat. No. 5,614,617; Froehler et al., U.S. Pat. No. 5,645,985; Cook etal., U.S. Pat. No. 5,681,941; Cook et al., U.S. Pat. No. 5,811,534; Cooket al., U.S. Pat. No. 5,750,692; Cook et al., U.S. Pat. No. 5,948,903;Cook et al., U.S. Pat. No. 5,587,470; Cook et al., U.S. Pat. No.5,457,191; Matteucci et al., U.S. Pat. No. 5,763,588; Froehler et al.,U.S. Pat. No. 5,830,653; Cook et al., U.S. Pat. No. 5,808,027; Cook etal., U.S. Pat. No. 6,166,199; and Matteucci et al., U.S. Pat. No.6,005,096.

Certain Modified Internucleoside Linkages

In certain embodiments, nucleosides of modified oligonucleotides may belinked together using any internucleoside linkage. The two main classesof internucleoside linking groups are defined by the presence or absenceof a phosphorus atom. Representative phosphorus-containinginternucleoside linkages include but are not limited to phosphodiesters,which contain a phosphodiester bond, P(O₂)═O, (also referred to asunmodified or naturally occurring linkages); phosphotriesters;methylphosphonates; methoxypropylphosphonates (“MOP”); phosphoramidates;mesyl phosphoramidates; phosphorothioates (P(O₂)═S); andphosphorodithioates (HS—P═S). Representative non-phosphorus containinginternucleoside linking groups include but are not limited tomethylenemethylimino (—CH₂—N(CH₃)—O—CH₂—); thiodiester, thionocarbamate(—O—C(═O)(NH)—S—); siloxane (—O—SiH₂—O—); and N,N′-dimethylhydrazine(—CH₂—N(CH₃)—N(CH₃)—). Modified internucleoside linkages, compared tonaturally occurring phosphate linkages, can be used to alter, typicallyincrease, nuclease resistance of the oligonucleotide. In certainembodiments, internucleoside linkages having a chiral atom can beprepared as a racemic mixture, or as separate enantiomers. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinginternucleoside linkages are well known to those skilled in the art.

Representative internucleoside linkages having a chiral center includebut are not limited to alkylphosphonates and phosphorothioates. Modifiedoligonucleotides comprising internucleoside linkages having a chiralcenter can be prepared as populations of modified oligonucleotidescomprising stereorandom internucleoside linkages, or as populations ofmodified oligonucleotides comprising phosphorothioate internucleosidelinkages in particular stereochemical configurations. In certainembodiments, populations of modified oligonucleotides comprisephosphorothioate internucleoside linkages wherein all of thephosphorothioate internucleoside linkages are stereorandom. Suchmodified oligonucleotides can be generated using synthetic methods thatresult in random selection of the stereochemical configuration of eachphosphorothioate internucleoside linkage. Nonetheless, as is wellunderstood by those of skill in the art, each individualphosphorothioate of each individual oligonucleotide molecule has adefined stereoconfiguration. In certain embodiments, populations ofmodified oligonucleotides are enriched for modified oligonucleotidescomprising one or more particular phosphorothioate internucleosidelinkages in a particular, independently selected stereochemicalconfiguration. In certain embodiments, the particular configuration ofthe particular phosphorothioate internucleoside linkage is present in atleast 65% of the molecules in the population. In certain embodiments,the particular configuration of the particular phosphorothioateinternucleoside linkage is present in at least 70% of the molecules inthe population. In certain embodiments, the particular configuration ofthe particular phosphorothioate internucleoside linkage is present in atleast 80% of the molecules in the population. In certain embodiments,the particular configuration of the particular phosphorothioateinternucleoside linkage is present in at least 90% of the molecules inthe population. In certain embodiments, the particular configuration ofthe particular phosphorothioate internucleoside linkage is present in atleast 99% of the molecules in the population. Such chirally enrichedpopulations of modified oligonucleotides can be generated usingsynthetic methods known in the art, e.g., methods described in Oka etal., JACS, 2003, 125, 8307, Wan et al. Nuc. Acid. Res., 2014, 42, 13456,and WO 2017/015555. In certain embodiments, a population of modifiedoligonucleotides is enriched for modified oligonucleotides having atleast one indicated phosphorothioate in the (Sp) configuration. Incertain embodiments, a population of modified oligonucleotides isenriched for modified oligonucleotides having at least onephosphorothioate in the (Rp) configuration. In certain embodiments,modified oligonucleotides comprising (Rp) and/or (Sp) phosphorothioatescomprise one or more of the following formulas, respectively, wherein“B” indicates a nucleobase:

Unless otherwise indicated, chiral internucleoside linkages of modifiedoligonucleotides described herein can be stereorandom or in a particularstereochemical configuration.

In certain embodiments, modified oligonucleotides comprise aninternucleoside motif of (5′ to 3′) sooosssssssssssssss. In certainembodiments, the particular stereochemical configuration of the modifiedoligonucleotides is (5′ to 3′)Sp-o-o-o-Sp-Sp-Sp-Rp-Sp-Sp-Rp-Sp-Sp-Sp-Sp-Sp-Sp-Sp-Sp orSp-o-o-o-Sp-Sp-Sp-Rp-Sp-Sp-Sp-Sp-Sp-Sp-Sp-Sp-Sp-Sp-Sp; wherein each ‘Sp’represents a phosphorothioate internucleoside linkage in the Sconfiguration; Rp represents a phosphorothioate internucleoside linkagein the R configuration; and ‘o’ represents a phosphodiesterinternucleoside linkage.

Neutral internucleoside linkages include, without limitation,phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), methoxypropyl, and thioformacetal (3′-S—CH₂—O-5′).Further neutral internucleoside linkages include nonionic linkagescomprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide,sulfide, sulfonate ester and amides (see e.g., CarbohydrateModifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds.,ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutralinternucleoside linkages include nonionic linkages comprising mixed N,O, S and CH₂ component parts.

In certain embodiments, a modified internucleoside linkage is any ofthose described in WO 2021/030778, incorporated by reference herein.

Certain Motifs

In certain embodiments, modified oligonucleotides comprise one or moremodified nucleosides comprising a modified sugar moiety. In certainembodiments, modified oligonucleotides comprise one or more modifiednucleosides comprising a modified nucleobase. In certain embodiments,modified oligonucleotides comprise one or more modified internucleosidelinkages. In such embodiments, the modified, unmodified, and differentlymodified sugar moieties, nucleobases, and/or internucleoside linkages ofa modified oligonucleotide define a pattern or motif. In certainembodiments, the patterns of sugar moieties, nucleobases, andinternucleoside linkages are each independent of one another. Thus, amodified oligonucleotide may be described by its sugar motif, nucleobasemotif and/or internucleoside linkage motif (as used herein, nucleobasemotif describes the modifications to the nucleobases independent of thesequence of nucleobases).

Certain Sugar Motifs

In certain embodiments, oligonucleotides comprise one or more type ofmodified sugar and/or unmodified sugar moiety arranged along theoligonucleotide, or portion thereof, in a defined pattern or sugarmotif. In certain instances, such sugar motifs include but are notlimited to any of the sugar modifications discussed herein.

In certain embodiments, modified oligonucleotides have a gapmer motif,which is defined by two external regions or “wings” and a central orinternal region or “gap.” The three regions of a gapmer motif (the5′-wing, the gap, and the 3′-wing) form a contiguous sequence ofnucleosides wherein at least some of the sugar moieties of thenucleosides of each of the wings differ from at least some of the sugarmoieties of the nucleosides of the gap. Specifically, at least the sugarmoieties of the nucleosides of each wing that are closest to the gap(the 3′-most nucleoside of the 5′-wing and the 5′-most nucleoside of the3′-wing) differ from the sugar moiety of the neighboring gapnucleosides, thus defining the boundary between the wings and the gap(i.e., the wing/gap junction). In certain embodiments, the sugarmoieties within the gap are the same as one another. In certainembodiments, the gap includes one or more nucleoside having a sugarmoiety that differs from the sugar moiety of one or more othernucleosides of the gap. In certain embodiments, the sugar motifs of thetwo wings are the same as one another (symmetric gapmer). In certainembodiments, the sugar motif of the 5′-wing differs from the sugar motifof the 3′-wing (asymmetric gapmer).

In certain embodiments, the wings of a gapmer comprise 1-6 nucleosides.In certain embodiments, each nucleoside of each wing of a gapmercomprises a modified sugar moiety. In certain embodiments, at least one,at least two, at least three, at least four, at least five, or at leastsix nucleosides of each wing of a gapmer comprises a modified sugarmoiety.

In certain embodiments, the gap of a gapmer comprises 7-12 nucleosides.In certain embodiments, each nucleoside of the gap of a gapmer comprisesa 2′-deoxyribosyl sugar moiety. In certain embodiments, at least onenucleoside of the gap of a gapmer comprises a modified sugar moiety andeach remaining nucleoside comprises a 2′-deoxyribosyl sugar moiety.

Herein, the lengths (number of nucleosides) of the three regions of agapmer may be provided using the notation [# of nucleosides in the5′-wing]-[# of nucleosides in the gap]-[# of nucleosides in the3′-wing]. Thus, a 5-10-5 gapmer consists of 5 linked nucleosides in eachwing and 10 linked nucleosides in the gap. Where such nomenclature isfollowed by a specific modification, that modification is themodification in each sugar moiety of each wing and the gap nucleosidescomprise a 2′-deoxyribosyl sugar moiety. Thus, a 5-10-5 MOE gapmerconsists of 5 linked 2′-MOE nucleosides in the 5′-wing, 10 linked2′-deoxyribonucleosides in the gap, and 5 linked 2′-MOE nucleosides inthe 3′-wing.

In certain embodiments, each nucleoside of a modified oligonucleotide,or portion thereof, comprises a 2′-substituted sugar moiety, a bicyclicsugar moiety, a sugar surrogate, or a 2′-deoxyribosyl sugar moiety. Incertain embodiments, the 2′-substituted sugar moiety is selected from a2′-MOE sugar moiety, a 2′-NMA sugar moiety, a 2′-OMe sugar moiety, and a2′-F sugar moiety. In certain embodiments, the bicyclic sugar moiety isselected from a cEt sugar moiety and an LNA sugar moiety. In certainembodiments, the sugar surrogate is selected from morpholino, modifiedmorpholino, PNA, THP, and F-HNA.

In certain embodiments, modified oligonucleotides comprise at least 12,at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, or at least 20 nucleosides comprising a modifiedsugar moiety. In certain embodiments, the modified sugar moiety isselected independently from a 2′-substituted sugar moiety, a bicyclicsugar moiety, or a sugar surrogate. In certain embodiments, the2′-substituted sugar moiety is selected from a 2′-MOE sugar moiety, a2′-NMA sugar moiety, a 2′-OMe sugar moiety, and a 2′-F sugar moiety. Incertain embodiments, the bicyclic sugar moiety is selected from a cEtsugar moiety and an LNA sugar moiety. In certain embodiments, the sugarsurrogate is selected from morpholino, modified morpholino, THP, andF-HNA.

In certain embodiments, each nucleoside of a modified oligonucleotidecomprises a modified sugar moiety (“fully modified oligonucleotide”). Incertain embodiments, each nucleoside of a fully modified oligonucleotidecomprises a 2′-substituted sugar moiety, a bicyclic sugar moiety, or asugar surrogate. In certain embodiments, the 2′-substituted sugar moietyis selected from a 2′-MOE sugar moiety, a 2′-NMA sugar moiety, a 2′-OMesugar moiety, and a 2′-F sugar moiety. In certain embodiments, thebicyclic sugar moiety is selected from a cEt sugar moiety and an LNAsugar moiety. In certain embodiments, the sugar surrogate is selectedfrom morpholino, modified morpholino, THP, and F-HNA. In certainembodiments, each nucleoside of a fully modified oligonucleotidecomprises the same modified sugar moiety (“uniformly modified sugarmotif”). In certain embodiments, the uniformly modified sugar motif is 7to 20 nucleosides in length. In certain embodiments, each nucleoside ofthe uniformly modified sugar motif comprises a 2′-substituted sugarmoiety, a bicyclic sugar moiety, or a sugar surrogate. In certainembodiments, the 2′-substituted sugar moiety is selected from a 2′-MOEsugar moiety, a 2′-NMA sugar moiety, a 2′-OMe sugar moiety, and a 2′-Fsugar moiety. In certain embodiments, the bicyclic sugar moiety isselected from a cEt sugar moiety and an LNA sugar moiety. In certainembodiments, the sugar surrogate is selected from morpholino, modifiedmorpholino, THP, and F-HNA. In certain embodiments, modifiedoligonucleotides having at least one fully modified sugar motif may alsocomprise at least 1, at least 2, at least 3, or at least 42′-deoxyribonucleosides.

Certain Nucleobase Motifs

In certain embodiments, oligonucleotides comprise modified and/orunmodified nucleobases arranged along the oligonucleotide, or portionthereof, in a defined pattern or motif. In certain embodiments, eachnucleobase is modified. In certain embodiments, none of the nucleobasesare modified. In certain embodiments, each purine or each pyrimidine ismodified. In certain embodiments, each adenine is modified. In certainembodiments, each guanine is modified. In certain embodiments, eachthymine is modified. In certain embodiments, each uracil is modified. Incertain embodiments, each cytosine is modified. In certain embodiments,some or all of the cytosine nucleobases in a modified oligonucleotideare 5-methyl cytosines. In certain embodiments, all of the cytosinenucleobases are 5-methyl cytosines and all of the other nucleobases ofthe modified oligonucleotide are unmodified nucleobases.

In certain embodiments, modified oligonucleotides comprise a block ofmodified nucleobases. In certain such embodiments, the block is at the3′-end of the oligonucleotide. In certain embodiments the block iswithin 3 nucleosides of the 3′-end of the oligonucleotide. In certainembodiments, the block is at the 5′-end of the oligonucleotide. Incertain embodiments the block is within 3 nucleosides of the 5′-end ofthe oligonucleotide.In certain embodiments, oligonucleotides having a gapmer motif comprisea nucleoside comprising a modified nucleobase. In certain suchembodiments, one nucleoside comprising a modified nucleobase is in thecentral gap of an oligonucleotide having a gapmer motif. In certain suchembodiments, the sugar moiety of the nucleoside is a 2′-deoxyribosylsugar moiety. In certain embodiments, the modified nucleobase isselected from: a 2-thiopyrimidine and a 5-propynepyrimidine.

Certain Internucleoside Linkage Motifs

In certain embodiments, oligonucleotides comprise modified and/orunmodified internucleoside linkages arranged along the oligonucleotide,or portion thereof, in a defined pattern or motif. In certainembodiments, each internucleoside linking group is a phosphodiesterinternucleoside linkage. In certain embodiments, each internucleosidelinking group of a modified oligonucleotide is a phosphorothioateinternucleoside linkage. In certain embodiments, each internucleosidelinkage of a modified oligonucleotide is independently selected from aphosphorothioate internucleoside linkage and phosphodiesterinternucleoside linkage. In certain embodiments, each phosphorothioateinternucleoside linkage is independently selected from a stereorandomphosphorothioate, a (Sp) phosphorothioate, and a (Rp) phosphorothioate.In certain embodiments, the sugar motif of a modified oligonucleotide isa gapmer and the internucleoside linkages within the gap are allmodified. In certain such embodiments, some or all of theinternucleoside linkages in the wings are unmodified phosphodiesterinternucleoside linkages. In certain embodiments, the terminalinternucleoside linkages are modified. In certain embodiments, the sugarmotif of a modified oligonucleotide is a gapmer, and the internucleosidelinkage motif comprises at least one phosphodiester internucleosidelinkage in at least one wing, wherein the at least one phosphodiesterinternucleoside linkage is not a terminal internucleoside linkage, andthe remaining internucleoside linkages are phosphorothioateinternucleoside linkages. In certain such embodiments, all of thephosphorothioate internucleoside linkages are stereorandom. In certainembodiments, all of the phosphorothioate internucleoside linkages in thewings are (Sp) phosphorothioates, and the gap comprises at least one Sp,Sp, Rp motif. In certain embodiments, populations of modifiedoligonucleotides are enriched for modified oligonucleotides comprisingsuch internucleoside linkage motifs. In certain embodiments, one or moreinternucleoside linkage is a mesyl phosphoramidate internucleosidelinkage. In certain embodiments, each internucleoside linkage isindependently selected from a phosphodiester internucleoside linkage, aphosphorothioate internucleoside linkage, and a mesyl phosphoramidateinternucleoside linkage. In certain embodiments, each internucleosidelinkage is independently selected from a phosphorothioateinternucleoside linkage and a mesyl phosphoramidate internucleosidelinkage. In certain embodiments, one or more internucleoside linkage isa methoxypropylphosphonate internucleoside linkage. In certainembodiments, each internucleoside linkage is independently selected froma phosphodiester internucleoside linkage, a phosphorothioateinternucleoside linkage, and a methoxypropylphosphonate internucleosidelinkage. In certain embodiments, each internucleoside linkage isindependently selected from a phosphorothioate internucleoside linkageand a methoxypropylphosphonate internucleoside linkage.

In certain embodiments, modified oligonucleotides comprise at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 16, at least 17, at least 18, orat least 19 phosphodiester internucleoside linkages. In certainembodiments, modified oligonucleotides comprise at least 1, at least 2,at least 3, at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, or at least 19phosphorothioate internucleoside linkages. In certain embodiments,modified oligonucleotides comprise at least 1, at least 2, at least 3,at least 4, or at least 5 phosphodiester internucleoside linkages andthe remainder of the internucleoside linkages are phosphorothioateinternucleoside linkages.

Certain Lengths

It is possible to increase or decrease the length of an oligonucleotidewithout eliminating activity. For example, in Woolf et al., Proc. Natl.Acad. Sci. USA, 1992, 89, 7305-7309, 1992), a series of oligonucleotides13-25 nucleobases in length were tested for their ability to inducecleavage of a target nucleic acid in an oocyte injection model.Oligonucleotides 25 nucleobases in length with 8 or 11 mismatch basesnear the ends of the oligonucleotides were able to direct specificcleavage of the target nucleic acid, albeit to a lesser extent than theoligonucleotides that contained no mismatches. Similarly, targetspecific cleavage was achieved using 13 nucleobase oligonucleotides,including those with 1 or 3 mismatches.

In certain embodiments, oligonucleotides (including modifiedoligonucleotides) can have any of a variety of ranges of lengths. Incertain embodiments, oligonucleotides consist of X to Y linkednucleosides, where X represents the fewest number of nucleosides in therange and Y represents the largest number nucleosides in the range. Incertain such embodiments, X and Y are each independently selected from8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, and 50; provided that X≤Y. For example, incertain embodiments, oligonucleotides consist of 12 to 13, 12 to 14, 12to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to30 linked nucleosides.

In certain embodiments, oligonucleotides consist of 16 linkednucleosides. In certain embodiments, oligonucleotides consist of 17linked nucleosides. In certain embodiments, oligonucleotides consist of18 linked nucleosides. In certain embodiments, oligonucleotides consistof 19 linked nucleosides. In certain embodiments, oligonucleotidesconsist of 20 linked nucleosides.

Certain Modified Oligonucleotides

In certain embodiments, the above modifications (sugar, nucleobase,internucleoside linkage) are incorporated into a modifiedoligonucleotide. In certain embodiments, modified oligonucleotides arecharacterized by their modification motifs and overall lengths. Incertain embodiments, such parameters are each independent of oneanother. Thus, unless otherwise indicated, each internucleoside linkageof an oligonucleotide having a gapmer sugar motif may be modified orunmodified and may or may not follow the gapmer modification pattern ofthe sugar modifications. For example, the internucleoside linkageswithin the wing regions of a sugar gapmer may be the same or differentfrom one another and may be the same or different from theinternucleoside linkages of the gap region of the sugar motif. Likewise,such sugar gapmer oligonucleotides may comprise one or more modifiednucleobase independent of the gapmer pattern of the sugar modifications.Unless otherwise indicated, all modifications are independent ofnucleobase sequence.

Certain Populations of Modified Oligonucleotides

Populations of modified oligonucleotides in which all of the modifiedoligonucleotides of the population have the same molecular formula canbe stereorandom populations or chirally enriched populations. All of thechiral centers of all of the modified oligonucleotides are stereorandomin a stereorandom population. In a chirally enriched population, atleast one particular chiral center is not stereorandom in the modifiedoligonucleotides of the population. In certain embodiments, the modifiedoligonucleotides of a chirally enriched population are enriched for β-Dribosyl sugar moieties, and all of the phosphorothioate internucleosidelinkages are stereorandom. In certain embodiments, the modifiedoligonucleotides of a chirally enriched population are enriched for bothβ-D ribosyl sugar moieties and at least one, particular phosphorothioateinternucleoside linkage in a particular stereochemical configuration.

Nucleobase Sequence

In certain embodiments, oligonucleotides (unmodified or modifiedoligonucleotides) are further described by their nucleobase sequence. Incertain embodiments oligonucleotides have a nucleobase sequence that iscomplementary to a second oligonucleotide or an identified referencenucleic acid, such as a target nucleic acid. In certain suchembodiments, a portion of an oligonucleotide has a nucleobase sequencethat is complementary to a second oligonucleotide or an identifiedreference nucleic acid, such as a target nucleic acid. In certainembodiments, the nucleobase sequence of a portion or entire length of anoligonucleotide is at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, or 100% complementary tothe second oligonucleotide or nucleic acid, such as a target nucleicacid.

Certain Oligomeric Compounds

In certain embodiments, provided herein are oligomeric compounds, whichconsist of an oligonucleotide (modified or unmodified) and optionallyone or more conjugate groups and/or terminal groups. Conjugate groupsconsist of one or more conjugate moiety and a conjugate linker whichlinks the conjugate moiety to the oligonucleotide. Conjugate groups maybe attached to either or both ends of an oligonucleotide and/or at anyinternal position. In certain embodiments, conjugate groups are attachedto the 2′-position of a nucleoside of a modified oligonucleotide. Incertain embodiments, conjugate groups that are attached to either orboth ends of an oligonucleotide are terminal groups. In certain suchembodiments, conjugate groups or terminal groups are attached at the 3′and/or 5′-end of oligonucleotides. In certain such embodiments,conjugate groups (or terminal groups) are attached at the 3′-end ofoligonucleotides. In certain embodiments, conjugate groups are attachednear the 3′-end of oligonucleotides. In certain embodiments, conjugategroups (or terminal groups) are attached at the 5′-end ofoligonucleotides. In certain embodiments, conjugate groups are attachednear the 5′-end of oligonucleotides.

Examples of terminal groups include but are not limited to conjugategroups, capping groups, phosphate moieties, protecting groups, abasicnucleosides, modified or unmodified nucleosides, and two or morenucleosides that are independently modified or unmodified.

Certain Conjugate Groups

In certain embodiments, oligonucleotides are covalently attached to oneor more conjugate groups. In certain embodiments, conjugate groupsmodify one or more properties of the attached oligonucleotide, includingbut not limited to pharmacodynamics, pharmacokinetics, stability,binding, absorption, tissue distribution, cellular distribution,cellular uptake, charge, and clearance. In certain embodiments,conjugate groups impart a new property on the attached oligonucleotide,e.g., fluorophores or reporter groups that enable detection of theoligonucleotide. Certain conjugate groups and conjugate moieties havebeen described previously, for example: cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid(Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett.,1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nuci. AcidsRes., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol orundecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118;Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al.,Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), an octadecylamine orhexylamino-calbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937), a tocopherol group (Nishina et al.,Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al.,Molecular Therapy, 2008, 16, 734-740), or a GalNAc cluster (e.g.,WO2014/179620).

Conjugate Moieties

Conjugate moieties include, without limitation, intercalators, reportermolecules, polyamines, polyamides, peptides, carbohydrates, vitaminmoieties, polyethylene glycols, thioethers, polyethers, cholesterols,thiocholesterols, cholic acid moieties, folate, lipids, lipophilicgroups, phospholipids, biotin, phenazine, phenanthridine, anthraquinone,adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores,and dyes.

In certain embodiments, a conjugate moiety comprises an active drugsubstance, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid,folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, anantidiabetic, an antibacterial, or an antibiotic.

Conjugate Linkers

Conjugate moieties are attached to oligonucleotides through conjugatelinkers. In certain oligomeric compounds, the conjugate linker is asingle chemical bond (i.e., the conjugate moiety is attached directly toan oligonucleotide through a single bond). In certain oligomericcompounds, a conjugate moiety is attached to an oligonucleotide via amore complex conjugate linker comprising one or more conjugate linkermoieties, which are sub-units making up a conjugate linker. In certainembodiments, the conjugate linker comprises a chain structure, such as ahydrocarbyl chain, or an oligomer of repeating units such as ethyleneglycol, nucleosides, or amino acid units.

In certain embodiments, a conjugate linker comprises one or more groupsselected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol,ether, thioether, and hydroxylamino. In certain such embodiments, theconjugate linker comprises groups selected from alkyl, amino, oxo, amideand ether groups. In certain embodiments, the conjugate linker comprisesgroups selected from alkyl and amide groups. In certain embodiments, theconjugate linker comprises groups selected from alkyl and ether groups.In certain embodiments, the conjugate linker comprises at least onephosphorus moiety. In certain embodiments, the conjugate linkercomprises at least one phosphate group. In certain embodiments, theconjugate linker includes at least one neutral linking group.

In certain embodiments, conjugate linkers, including the conjugatelinkers described above, are bifunctional linking moieties, e.g., thoseknown in the art to be useful for attaching conjugate groups to parentcompounds, such as the oligonucleotides provided herein. In general, abifunctional linking moiety comprises at least two functional groups.One of the functional groups is selected to bind to a particular site ona parent compound and the other is selected to bind to a conjugategroup. Examples of functional groups used in a bifunctional linkingmoiety include but are not limited to electrophiles for reacting withnucleophilic groups and nucleophiles for reacting with electrophilicgroups. In certain embodiments, bifunctional linking moieties compriseone or more groups selected from amino, hydroxyl, carboxylic acid,thiol, alkyl, alkenyl, and alkynyl.

Examples of conjugate linkers include but are not limited topyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-calboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include butare not limited to substituted or unsubstituted C₁-C₁₀ alkyl,substituted or unsubstituted C₂-C₁₀ alkenyl or substituted orunsubstituted C₂-C₁₀ alkynyl, wherein a nonlimiting list of preferredsubstituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl andalkynyl.

In certain embodiments, conjugate linkers comprise 1-10linker-nucleosides. In certain embodiments, conjugate linkers comprise2-5 linker-nucleosides. In certain embodiments, conjugate linkerscomprise exactly 3 linker-nucleosides. In certain embodiments, conjugatelinkers comprise the TCA motif. In certain embodiments, suchlinker-nucleosides are modified nucleosides. In certain embodiments suchlinker-nucleosides comprise a modified sugar moiety. In certainembodiments, linker-nucleosides are unmodified. In certain embodiments,linker-nucleosides comprise an optionally protected heterocyclic baseselected from a purine, substituted purine, pyrimidine or substitutedpyrimidine. In certain embodiments, a cleavable moiety is a nucleosideselected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methyl cytosine, adenine, 6-N-benzoyladenine,guanine and 2-N-isobutyrylguanine. It is typically desirable forlinker-nucleosides to be cleaved from the oligomeric compound after itreaches a target tissue. Accordingly, linker-nucleosides are typicallylinked to one another and to the remainder of the oligomeric compoundthrough cleavable bonds. In certain embodiments, such cleavable bondsare phosphodiester bonds.

Herein, linker-nucleosides are not considered to be part of theoligonucleotide. Accordingly, in embodiments in which an oligomericcompound comprises an oligonucleotide consisting of a specified numberor range of linked nucleosides and/or a specified percentcomplementarity to a reference nucleic acid and the oligomeric compoundalso comprises a conjugate group comprising a conjugate linkercomprising linker-nucleosides, those linker-nucleosides are not countedtoward the length of the oligonucleotide and are not used in determiningthe percent complementarity of the oligonucleotide for the referencenucleic acid. For example, an oligomeric compound may comprise (1) amodified oligonucleotide consisting of 8-30 nucleosides and (2) aconjugate group comprising 1-10 linker-nucleosides that are contiguouswith the nucleosides of the modified oligonucleotide. The total numberof contiguous linked nucleosides in such an oligomeric compound is morethan 30. Alternatively, an oligomeric compound may comprise a modifiedoligonucleotide consisting of 8-30 nucleosides and no conjugate group.The total number of contiguous linked nucleosides in such an oligomericcompound is no more than 30. Unless otherwise indicated conjugatelinkers comprise no more than 10 linker-nucleosides. In certainembodiments, conjugate linkers comprise no more than 5linker-nucleosides. In certain embodiments, conjugate linkers compriseno more than 3 linker-nucleosides. In certain embodiments, conjugatelinkers comprise no more than 2 linker-nucleosides. In certainembodiments, conjugate linkers comprise no more than 1linker-nucleoside.

In certain embodiments, it is desirable for a conjugate group to becleaved from the oligonucleotide. For example, in certain circumstancesoligomeric compounds comprising a particular conjugate moiety are bettertaken up by a particular cell type, but once the oligomeric compound hasbeen taken up, it is desirable that the conjugate group be cleaved torelease the unconjugated or parent oligonucleotide. Thus, certainconjugate linkers may comprise one or more cleavable moieties. Incertain embodiments, a cleavable moiety is a cleavable bond. In certainembodiments, a cleavable moiety is a group of atoms comprising at leastone cleavable bond. In certain embodiments, a cleavable moiety comprisesa group of atoms having one, two, three, four, or more than fourcleavable bonds. In certain embodiments, a cleavable moiety isselectively cleaved inside a cell or subcellular compartment, such as alysosome. In certain embodiments, a cleavable moiety is selectivelycleaved by endogenous enzymes, such as nucleases.

In certain embodiments, a cleavable bond is selected from among: anamide, an ester, an ether, one or both esters of a phosphodiester, aphosphate ester, a carbamate, or a disulfide. In certain embodiments, acleavable bond is one or both of the esters of a phosphodiester. Incertain embodiments, a cleavable moiety comprises a phosphate orphosphodiester. In certain embodiments, the cleavable moiety is aphosphate linkage between an oligonucleotide and a conjugate moiety orconjugate group.

In certain embodiments, a cleavable moiety comprises or consists of oneor more linker-nucleosides. In certain such embodiments, the one or morelinker-nucleosides are linked to one another and/or to the remainder ofthe oligomeric compound through cleavable bonds. In certain embodiments,such cleavable bonds are unmodified phosphodiester bonds. In certainembodiments, a cleavable moiety is 2′-deoxyribonucleoside that isattached to either the 3′ or 5′-terminal nucleoside of anoligonucleotide by a phosphate internucleoside linkage and covalentlyattached to the remainder of the conjugate linker or conjugate moiety bya phosphate or phosphorothioate internucleoside linkage. In certain suchembodiments, the cleavable moiety is 2′-deoxyadenosine.

Certain Terminal Groups

In certain embodiments, oligomeric compounds comprise one or moreterminal groups. In certain such embodiments, oligomeric compoundscomprise a stabilized 5′-phosphate. Stabilized 5′-phosphates include,but are not limited to 5′-phosphanates, including, but not limited to5′-vinylphosphonates. In certain embodiments, terminal groups compriseone or more abasic nucleosides and/or inverted nucleosides. In certainembodiments, terminal groups comprise one or more 2′-linked nucleosides.In certain such embodiments, the 2′-linked nucleoside is an abasicnucleoside.

Oligomeric Duplexes

In certain embodiments, oligomeric compounds described herein comprisean oligonucleotide, having a nucleobase sequence complementary to thatof a target nucleic acid. In certain embodiments, an oligomeric compoundis paired with a second oligomeric compound to form an oligomericduplex. Such oligomeric duplexes comprise a first oligomeric compoundhaving a portion complementary to a target nucleic acid and a secondoligomeric compound having a portion complementary to the firstoligomeric compound. In certain embodiments, the first oligomericcompound of an oligomeric duplex comprises or consists of (1) a modifiedor unmodified oligonucleotide and optionally a conjugate group and (2) asecond modified or unmodified oligonucleotide and optionally a conjugategroup. Either or both oligomeric compounds of an oligomeric duplex maycomprise a conjugate group. The oligonucleotides of each oligomericcompound of an oligomeric duplex may include non-complementaryoverhanging nucleosides.

Antisense Activity

In certain embodiments, oligomeric compounds and oligomeric duplexes arecapable of hybridizing to a target nucleic acid, resulting in at leastone antisense activity; such oligomeric compounds and oligomericduplexes are antisense compounds. In certain embodiments, antisensecompounds have antisense activity when they reduce, modulate, orincrease the amount or activity of a target nucleic acid by 25% or morein the standard cell assay. In certain embodiments, antisense compoundsselectively affect one or more target nucleic acid. Such antisensecompounds comprise a nucleobase sequence that hybridizes to one or moretarget nucleic acid, resulting in one or more desired antisense activityand does not hybridize to one or more non-target nucleic acid or doesnot hybridize to one or more non-target nucleic acid in such a way thatresults in significant undesired antisense activity.

In certain antisense activities, hybridization of an antisense compoundto a target nucleic acid results in recruitment of a protein thatcleaves the target nucleic acid. For example, certain antisensecompounds result in RNase H mediated cleavage of the target nucleicacid. RNase H is a cellular endonuclease that cleaves the RNA strand ofan RNA:DNA duplex. The DNA in such an RNA:DNA duplex need not beunmodified DNA. In certain embodiments, provided herein are antisensecompounds that are sufficiently “DNA-like” to elicit RNase H activity.In certain embodiments, one or more non-DNA-like nucleoside in the gapof a gapmer is tolerated.

In certain antisense activities, an antisense compound or a portion ofan antisense compound is loaded into an RNA-induced silencing complex(RISC), ultimately resulting in cleavage of the target nucleic acid. Forexample, certain antisense compounds result in cleavage of the targetnucleic acid by Argonaute Antisense compounds that are loaded into RISCare RNAi compounds. RNAi compounds may be double-stranded (siRNA) orsingle-stranded (ssRNA).

In certain embodiments, hybridization of an antisense compound to atarget nucleic acid does not result in recruitment of a protein thatcleaves that target nucleic acid. In certain embodiments, hybridizationof the antisense compound to the target nucleic acid results inalteration of splicing of the target nucleic acid. In certainembodiments, hybridization of an antisense compound to a target nucleicacid results in inhibition of a binding interaction between the targetnucleic acid and a protein or other nucleic acid. In certainembodiments, hybridization of an antisense compound to a target nucleicacid results in alteration of translation of the target nucleic acid. Incertain embodiments, hybridization of an antisense compound to a targetnucleic acid results in exon inclusion. In certain embodiments,hybridization of an antisense compound to a target nucleic acid resultsin an increase in the amount or activity of a target nucleic acid. Incertain embodiments, hybridization of an antisense compoundcomplementary to a target nucleic acid results in alteration ofsplicing, leading to the inclusion of an exon in the mRNA.

Antisense activities may be observed directly or indirectly. In certainembodiments, observation or detection of an antisense activity involvesobservation or detection of a change in an amount of a target nucleicacid or protein encoded by such target nucleic acid, a change in theratio of splice variants of a nucleic acid or protein and/or aphenotypic change in a cell or subject.

Certain Target Nucleic Acids

In certain embodiments, oligomeric compounds comprise or consist of anoligonucleotide comprising a portion that is complementary to a targetnucleic acid. In certain embodiments, the target nucleic acid is anendogenous RNA molecule. In certain embodiments, the target nucleic acidencodes a protein. In certain such embodiments, the target nucleic acidis selected from: a mature mRNA and a pre-mRNA, including intronic,exonic and untranslated regions. In certain embodiments, the targetnucleic acid is a mature mRNA. In certain embodiments, the targetnucleic acid is a pre-mRNA. In certain embodiments, the target region isentirely within an intron. In certain embodiments, the target regionspans an intron/exon junction. In certain embodiments, the target regionis at least 50% within an intron.

Complementarity/Mismatches to the Target Nucleic Acid

It is possible to introduce mismatch bases without eliminating activity.For example, Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March2001) demonstrated the ability of an oligonucleotide having 100%complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xLmRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and invivo. Furthermore, this oligonucleotide demonstrated potent anti-tumoractivity in vivo. Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988)tested a series of tandem 14 nucleobase oligonucleotides, and a 28 and42 nucleobase oligonucleotides comprised of the sequence of two or threeof the tandem oligonucleotides, respectively, for their ability toarrest translation of human DHFR in a rabbit reticulocyte assay. Each ofthe three 14 nucleobase oligonucleotides alone was able to inhibittranslation, albeit at a more modest level than the 28 or 42 nucleobaseoligonucleotides.

In certain embodiments, oligonucleotides are complementary to the targetnucleic acid over the entire length of the oligonucleotide. In certainembodiments, oligonucleotides are 99%, 95%, 90%, 85%, or 80%complementary to the target nucleic acid. In certain embodiments,oligonucleotides are at least 80% complementary to the target nucleicacid over the entire length of the oligonucleotide and comprise aportion that is 100% or fully complementary to a target nucleic acid. Incertain embodiments, the portion of full complementarity is 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length.

In certain embodiments, oligonucleotides comprise one or more mismatchednucleobases relative to the target nucleic acid. In certain embodiments,the mismatch is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 from the 5′-end of the oligonucleotide.

SMN2

In certain embodiments, oligomeric compounds comprise or consist of amodified oligonucleotide that is complementary to a target nucleic acidencoding SMN2, or a portion thereof. In certain embodiments, SMN2 hasthe sequence set forth in SEQ ID NO: 1 (GENBANK Accession No.NT_006713.14 truncated from nucleotides 19939708 to Ser. No.19/967,777).

In certain embodiments, contacting a cell with the oligomeric compoundcomplementary to SEQ ID NO: 1 modulates the splicing of SMN2 RNA in acell. In certain embodiments, contacting a cell with the oligomericcompound complementary to SEQ ID NO: 1 increases the amount of SMN2 RNAincluding exon 7. In certain embodiments, contacting a cell with theoligomeric compound complementary to SEQ ID NO: 1 increases full-lengthSMN2 protein expression. In certain embodiments, the oligomeric compoundconsists of a modified oligonucleotide.

In certain embodiments, contacting a cell in a subject with anoligomeric compound complementary to SEQ ID NO: 1 ameliorates one ormore symptom of a neurodegenerative disease. In certain embodiments, theneurodegenerative disease is SMA, including Type I SMA, Type II SMA,Type III SMA, and Type IV SMA. In certain embodiments, the symptom isany of reduced muscle strength; inability or reduced ability to situpright, to stand, and/or walk; reduced neuromuscular activity; reducedelectrical activity in one or more muscles; reduced respiration;inability or reduced ability to eat, drink, and/or breathe withoutassistance; loss of weight or reduced weight gain; and/or decreasedsurvival.

In certain embodiments, an oligomeric compound complementary to SEQ IDNO: 1 is capable of increasing SMN2 RNA including exon 7 in vivo by atleast 1 fold, 2 fold, or 3 fold when administered according to thestandard in vivo assay. In certain embodiments, an oligomeric compoundcomplementary to SEQ ID NO: 1 is capable of increasing full-length SMN2protein in vivo by at least 1 fold, 2 fold, or 3 fold when administeredaccording to the standard in vivo assay.

Certain Target Nucleic Acids in Certain Tissues

In certain embodiments, oligomeric compounds comprise or consist of anoligonucleotide comprising a portion that is complementary to a targetnucleic acid, wherein the target nucleic acid is expressed in apharmacologically relevant tissue. In certain embodiments, thepharmacologically relevant tissues are the cells and tissues thatcomprise the central nervous system (CNS). Such tissues include braintissues, such as, spinal cord, cortex, and coronal brain tissue.

Certain Pharmaceutical Compositions

In certain embodiments, described herein are pharmaceutical compositionscomprising one or more oligomeric compounds. In certain embodiments, theone or more oligomeric compounds each consists of a modifiedoligonucleotide. In certain embodiments, the pharmaceutical compositioncomprises a pharmaceutically acceptable diluent or carrier. In certainembodiments, a pharmaceutical composition comprises or consists of asterile saline solution and one or more oligomeric compound. In certainembodiments, the sterile saline is pharmaceutical grade saline. Incertain embodiments, a pharmaceutical composition comprises or consistsof one or more oligomeric compound and sterile water. In certainembodiments, the sterile water is pharmaceutical grade water. In certainembodiments, a pharmaceutical composition comprises or consists of oneor more oligomeric compound and phosphate-buffered saline (PBS). Incertain embodiments, the sterile PBS is pharmaceutical grade PBS. Incertain embodiments, a pharmaceutical composition comprises or consistsof one or more oligomeric compound and artificial cerebrospinal fluid(“artificial CSF” or “aCSF”). In certain embodiments, the artificialcerebrospinal fluid is pharmaceutical grade. In certain embodiments, apharmaceutical composition comprises a modified oligonucleotide andartificial cerebrospinal fluid. In certain embodiments, a pharmaceuticalcomposition consists of a modified oligonucleotide and artificialcerebrospinal fluid. In certain embodiments, a pharmaceuticalcomposition consists essentially of a modified oligonucleotide andartificial cerebrospinal fluid. In certain embodiments, the artificialcerebrospinal fluid is pharmaceutical grade.

In certain embodiments, pharmaceutical compositions comprise one or moreoligomeric compound and one or more excipients. In certain embodiments,excipients are selected from water, salt solutions, alcohol,polyethylene glycols, gelatin, lactose, amylase, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose andpolyvinylpyrrolidone.

In certain embodiments, oligomeric compounds may be admixed withpharmaceutically acceptable active and/or inert substances for thepreparation of pharmaceutical compositions or formulations. Compositionsand methods for the formulation of pharmaceutical compositions depend ona number of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

In certain embodiments, pharmaceutical compositions comprising anoligomeric compound encompass any pharmaceutically acceptable salts ofthe oligomeric compound, esters of the oligomeric compound, or salts ofsuch esters. In certain embodiments, pharmaceutical compositionscomprising oligomeric compounds comprising one or more oligonucleotide,upon administration to a subject, including a human, are capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto pharmaceutically acceptable salts of oligomeric compounds, prodrugs,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. Suitable pharmaceutically acceptable salts include, butare not limited to, sodium and potassium salts. In certain embodiments,prodrugs comprise one or more conjugate group attached to anoligonucleotide, wherein the conjugate group is cleaved by endogenousnucleases within the body. In certain embodiments, prodrugs comprise oneor more conjugate group attached to an oligonucleotide, wherein theconjugate group is cleaved by endogenous nucleases within the body.

Lipid moieties have been used in nucleic acid therapies in a variety ofmethods. In certain such methods, the nucleic acid, such as anoligomeric compound, is introduced into preformed liposomes orlipoplexes made of mixtures of cationic lipids and neutral lipids. Incertain methods, DNA complexes with mono- or poly-cationic lipids areformed without the presence of a neutral lipid. In certain embodiments,a lipid moiety is selected to increase distribution of a pharmaceuticalagent to a particular cell or tissue. In certain embodiments, a lipidmoiety is selected to increase distribution of a pharmaceutical agent tofat tissue. In certain embodiments, a lipid moiety is selected toincrease distribution of a pharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions comprise a deliverysystem. Examples of delivery systems include, but are not limited to,liposomes and emulsions. Certain delivery systems are useful forpreparing certain pharmaceutical compositions including those comprisinghydrophobic compounds. In certain embodiments, certain organic solventssuch as dimethylsulfoxide are used.

In certain embodiments, pharmaceutical compositions comprise one or moretissue-specific delivery molecules designed to deliver the one or morepharmaceutical agents comprising an oligomeric compound provided hereinto specific tissues or cell types. For example, in certain embodiments,pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, pharmaceutical compositions comprise aco-solvent system. Certain of such co-solvent systems comprise, forexample, benzyl alcohol, a nonpolar surfactant, a water-miscible organicpolymer, and an aqueous phase. In certain embodiments, such co-solventsystems are used for hydrophobic compounds. A non-limiting example ofsuch a co-solvent system is the VPD co-solvent system, which is asolution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v ofthe nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol300. The proportions of such co-solvent systems may be variedconsiderably without significantly altering their solubility andtoxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, pharmaceutical compositions are prepared fororal administration. In certain embodiments, pharmaceutical compositionsare prepared for buccal administration. In certain embodiments, apharmaceutical composition is prepared for administration by injection(e.g., intravenous, subcutaneous, intramuscular, intrathecal (IT),intracerebroventricular (ICV), etc.). In certain of such embodiments, apharmaceutical composition comprises a carrier and is formulated inaqueous solution, such as water or physiologically compatible bufferssuch as Hanks's solution, Ringer's solution, or physiological salinebuffer. In certain embodiments, other ingredients are included (e.g.,ingredients that aid in solubility or serve as preservatives). Incertain embodiments, injectable suspensions are prepared usingappropriate liquid carriers, suspending agents and the like. Certainpharmaceutical compositions for injection are presented in unit dosageform, e.g., in ampoules or in multi-dose containers. Certainpharmaceutical compositions for injection are suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents. Certainsolvents suitable for use in pharmaceutical compositions for injectioninclude, but are not limited to, lipophilic solvents and fatty oils,such as sesame oil, synthetic fatty acid esters, such as ethyl oleate ortriglycerides, and liposomes.

Under certain conditions, certain compounds disclosed herein act asacids. Although such compounds may be drawn or described in protonated(free acid) form, or ionized and in association with a cation (salt)form, aqueous solutions of such compounds exist in equilibrium amongsuch forms. For example, a phosphate linkage of an oligonucleotide inaqueous solution exists in equilibrium among free acid, anion and saltforms. Unless otherwise indicated, compounds described herein areintended to include all such forms. Moreover, certain oligonucleotideshave several such linkages, each of which is in equilibrium. Thus,oligonucleotides in solution exist in an ensemble of forms at multiplepositions all at equilibrium. The term “oligonucleotide” is intended toinclude all such forms. Drawn structures necessarily depict a singleform. Nevertheless, unless otherwise indicated, such drawings arelikewise intended to include corresponding forms. Herein, a structuredepicting the free acid of a compound followed by the term “or a saltthereof” expressly includes all such forms that may be fully orpartially protonated/de-protonated/in association with a cation. Incertain instances, one or more specific cation is identified.

In certain embodiments, modified oligonucleotides or oligomericcompounds are in aqueous solution with sodium. In certain embodiments,modified oligonucleotides or oligomeric compounds are in aqueoussolution with potassium. In certain embodiments, modifiedoligonucleotides or oligomeric compounds are in PBS. In certainembodiments, modified oligonucleotides or oligomeric compounds are inwater. In certain such embodiments, the pH of the solution is adjustedwith NaOH and/or HCl to achieve a desired pH.

Herein, certain specific doses are described. A dose may be in the formof a dosage unit. For clarity, a dose (or dosage unit) of a modifiedoligonucleotide or an oligomeric compound in milligrams indicates themass of the free acid form of the modified oligonucleotide or oligomericcompound. As described above, in aqueous solution, the free acid is inequilibrium with anionic and salt forms. However, for the purpose ofcalculating dose, it is assumed that the modified oligonucleotide oroligomeric compound exists as a solvent-free, sodium-acetate free,anhydrous, free acid. For example, where a modified oligonucleotide oran oligomeric compound is in solution comprising sodium (e.g., saline),the modified oligonucleotide or oligomeric compound may be partially orfully de-protonated and in association with Na⁺ ions. However, the massof the protons are nevertheless counted toward the weight of the dose,and the mass of the Na⁺ ions are not counted toward the weight of thedose. Thus, for example, a dose, or dosage unit, of 10 mg of CompoundNo. 1263789, Compound No. 1287717, Compound No. 1287745, and CompoundNo. 1358996 equals the number of fully protonated molecules that weighs10 mg. This would be equivalent to 10.53 mg of solvent-free, sodiumacetate-free, anhydrous sodiated Compound No. 1263789, 10.53 mg ofsolvent-free, sodium acetate-free, anhydrous sodiated Compound No.1287717, 10.52 mg of solvent-free, sodium acetate-free, anhydroussodiated Compound No. 1287745, and 10.51 mg of solvent-free, sodiumacetate-free, anhydrous sodiated Compound No. 1358996. When anoligomeric compound comprises a conjugate group, the mass of theconjugate group is included in calculating the dose of such oligomericcompound. If the conjugate group also has an acid, the conjugate groupis likewise assumed to be fully protonated for the purpose ofcalculating dose.

Certain Compositions Compound No: 1263789

In certain embodiments, Compound No. 1263789 is characterized as amodified oligonucleotide having a sequence of (from 5′ to 3′)CACTTTCATAATGCTGGC (SEQ ID NO: 21), wherein each nucleoside comprises a2′-MOE sugar moiety, wherein the internucleoside linkages betweennucleosides 2 to 3 and 4 to 5 are phosphodiester internucleosidelinkages and the internucleoside linkages between nucleosides 1 to 2, 3to 4, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, and 17 to 18 arephosphorothioate internucleoside linkages, and wherein each cytosine isa 5-methyl cytosine.

In certain embodiments, Compound No. 1263789 is represented by thefollowing chemical notation (5′ to 3′): ^(m)C_(ns) A_(no) ^(m)C_(ns)T_(no) T_(ns) T_(ns) ^(m)C_(ns) A_(ns) T_(ns) A_(ns) A_(ns) T_(ns)G_(ns) ^(m)C_(ns) T_(ns) G_(ns) G_(ns) ^(m)C_(n) (SEQ ID NO: 21),

wherein,A=an adenine nucleobase,^(m)C=a 5-methyl cytosine nucleobase,G=a guanine nucleobase,T=a thymine nucleobase,e=a 2′-MOE sugar moiety,s=a phosphorothioate internucleoside linkage, ando=a phosphodiester internucleoside linkage.

In certain embodiments, Compound No. 1263789 is represented by thefollowing chemical structure:

Structure 1. Compound No. 1263789

In certain embodiments, the sodium salt of Compound No. 1263789 isrepresented by the following chemical structure:

Structure 2. The sodium salt of Compound No. 1263789

Compound No: 1287717

In certain embodiments, Compound No. 1287717 is characterized as amodified oligonucleotide having a sequence of (from 5′ to 3′)TTCACTTTCATAATGCTGGC (SEQ ID NO: 22), wherein each nucleoside comprisesa 2′-MOE sugar moiety, wherein the internucleoside linkages betweennucleosides 1 to 2 and 19 to 20 are phosphodiester internucleosidelinkages and the internucleoside linkages between nucleosides 2 to 3, 3to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, 17 to 18, and 18to 19 are phosphorothioate internucleoside linkages, and wherein eachcytosine is a 5-methyl cytosine.

In certain embodiments, Compound No. 1287717 is represented by thefollowing chemical notation (5′ to 3′): T_(eo) T_(es) ^(m)C_(es) A_(es)^(m)C_(es) T_(es) T_(es) T_(es) ^(m)C_(es) A_(es) T_(es) A_(es) A_(es)T_(es) G_(es) ^(m)C_(es) T_(es) G_(es) G_(eo) ^(m)C_(e) (SEQ ID NO: 22)

wherein,A=an adenine nucleobase,^(m)C=a 5-methyl cytosine nucleobase,

G=a guanine nucleobase,

T=a thymine nucleobase,

e=a 2′-MOE sugar moiety,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

In certain embodiments, Compound No. 1287717 is represented by thefollowing chemical structure:

Structure 3. Compound No. 1287717

In certain embodiments, the sodium salt of Compound No. 1287717 isrepresented by the following chemical structure:

Structure 4. The sodium salt of Compound No. 1287717

Compound No: 1287745

In certain embodiments, Compound No. 1287745 is characterized as amodified oligonucleotide having a sequence of (from 5′ to 3′)TTCACTTTCATAATGCTGGC (SEQ ID NO: 22), wherein each of nucleosides 1 and20 comprises a 2′-MOE sugar moiety, each of nucleosides 2-19 comprises a2′-NMA sugar moiety, wherein the internucleoside linkages betweennucleosides 1 to 2 and 19 to 20 are phosphodiester internucleosidelinkages and the internucleoside linkages between nucleosides 2 to 3, 3to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, 17 to 18, and 18to 19 are phosphorothioate internucleoside linkages, and wherein eachcytosine is a 5-methyl cytosine.

In certain embodiments, Compound No. 1287745 is represented by thefollowing chemical notation (5′ to 3′): T_(eo) T_(es) ^(m)C_(es) A_(es)^(m)C_(es) T_(es) T_(es) T_(es) ^(m)C_(es) A_(es) T_(es) A_(es) A_(es)T_(es) G_(es) ^(m)C_(es) T_(es) G_(es) G_(eo) ^(m)C_(e) (SEQ ID NO: 22)

wherein,A=an adenine nucleobase,^(m)C=a 5-methyl cytosine nucleobase,G=a guanine nucleobase,T=a thymine nucleobase,e=a 2′-MOE sugar moiety,n=a 2′-NMA sugar moiety,s=a phosphorothioate internucleoside linkage, ando=a phosphodiester internucleoside linkage.

In certain embodiments, Compound No. 1287745 is represented by thefollowing chemical structure:

Structure 5. Compound No. 1287745

In certain embodiments, the sodium salt of Compound No. 1287745 isrepresented by the following chemical structure:

Structure 6. The sodium salt of Compound No. 1287745

Compound No: 1358996

In certain embodiments, Compound No. 1358996 is characterized asmodified oligonucleotide having a sequence of (from 5′ to 3′)CACTTTCATAATGCTGGC (SEQ ID NO: 21), wherein each nucleoside comprises a2′-NMA sugar moiety, wherein the internucleoside linkages betweennucleosides 2 to 3 and 4 to 5 are phosphodiester internucleosidelinkages and the internucleoside linkages between nucleosides 1 to 2, 3to 4, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, and 17 to 18 arephosphorothioate internucleoside linkages, and wherein each cytosine isa 5-methyl cytosine.

In certain embodiments, Compound No. 1358996 is represented by thefollowing chemical notation (5′ to 3′): ^(m)C_(ns) A_(no) ^(m)C_(ns)T_(no) T_(ns) T_(ns) ^(m)C_(ns) A_(ns) T_(ns) A_(ns) A_(ns) T_(ns)G_(ns) ^(m)C_(ns) T_(ns) G_(ns) G_(ns) ^(m)C_(n) (SEQ ID NO: 21)

wherein,A=an adenine nucleobase,^(m)C=a 5-methyl cytosine nucleobase,G=a guanine nucleobase,T=a thymine nucleobase,n=a 2′-NMA sugar moiety,s=a phosphorothioate internucleoside linkage, ando=a phosphodiester internucleoside linkage.

In certain embodiments, Compound No. 1358996 is represented by thefollowing chemical structure:

Structure 7. Compound No. 1358996

In certain embodiments, the sodium salt of Compound No. 1358996 isrepresented by the following chemical structure:

Structure 8. The Sodium Salt of Compound No. 1358996

Certain Comparator Compositions

In certain embodiments, Spinraza® (generic name nusinersen; Compound No.396443), approved for treatment of SMA, is a comparator compound (See,e.g., Chiroboga, et al., Neurology, 86(10): 890-897, 2016; Finkel, etal., Lancet, 338(10063): 3017-3026, 2016; Finkel, et al., N. Engl. J.Med., 377(18):1723-1732 2017; Mercuri, et al., N. Engl. J. Med.,378(7):625-635, 2018; Montes, et al., Muscle Nerve. 60(4): 409-414,2019; Darras, et al., Neurology, 92(21):e2492-e2506, 2019). Spinraza®was previously described in WO2010120820, incorporated herein byreference, and has a sequence (from 5′ to 3′) of TCACTTTCATAATGCTGG (SEQID NO: 23), wherein each nucleoside comprises a 2′-MOE sugar moiety,each internucleoside linkage is a phosphorothioate internucleosidelinkage, and each cytosine is a 5-methyl cytosine.

In certain embodiments, although not approved for human therapy, otherpreviously described compounds, including Compound Nos. 387954, 396442,443305, and 819735, are comparator compounds.

Compound No. 387954 was previously described in WO 2014/179620,incorporated herein by reference. Compound No. 387954 has a sequence(from 5′ to 3′) of ATTCACTTTCATAATGCTGG (SEQ ID NO: 20), wherein eachnucleoside comprises a 2′-MOE sugar moiety, each internucleoside linkageis a phosphorothioate internucleoside linkage, and each cytosine is a5-methyl cytosine.

Compound No. 396442 was previously described in WO 2010/120820,incorporated herein by reference. Compound No. 396442 has a sequence(from 5′ to 3′) of CACTTTCATAATGCTGGC (SEQ ID NO: 21), wherein eachnucleoside comprises a 2′-MOE sugar moiety, each internucleoside linkageis a phosphorothioate internucleoside linkage, and each cytosine is a5-methyl cytosine.

Compound No. 443305 was previously described in WO 2018/014041,incorporated herein by reference. Compound No. 443305 has a sequence(from 5′ to 3′) of TCACTTTCATAATGCTGG (SEQ ID NO: 23), wherein eachnucleoside comprises a 2′-NMA sugar moiety, each internucleoside linkageis a phosphorothioate internucleoside linkage, and each cytosine is a5-methyl cytosine.

Compound No. 819735 was previously described in WO 2018/014041,incorporated herein by reference. Compound No. 819735 has a sequence(from 5′ to 3′) of CACTTTCATAATGCTGGC (SEQ ID NO: 21), wherein eachnucleoside comprises a 2′-NMA sugar moiety, each internucleoside linkageis a phosphorothioate internucleoside linkage, and each cytosine is a5-methyl cytosine.

TABLE 1 Certain Comparator Compositions SEQ Compound Nucleobase SequenceInternucleoside ID Number (5′ to 3′) Sugar Motif Linkage Motif NO:Reference Number 396443 TCACTTTCATAATGCTGG Full 2′- MOE Full PS 23WO 2010/120820 387954 ATTCACTTTCATAATGCTGG Full 2′- MOE Full PS 20WO 2014/179620 396442 CACTTTCATAATGCTGGC Full 2′- MOE Full PS 21WO 2010/120820 443305 TCACTTTCATAATGCTGG Full 2′- NMA Full PS 23WO 2018/014041 819735 CACTTTCATAATGCTGGC Full 2′- NMA Full PS 21WO 2018/014041

In certain embodiments, compounds described herein are superior relativeto compounds described in WO 2007/002390, WO2010/120820, WO 2015/161170,and WO 2018/014041, because they demonstrate one or more improvedproperties, such as, potency, efficacy, and tolerability.

For example, Compound No. 1263789, Compound No. 1287745, and CompoundNo. 1358996 each demonstrated improved potency in vivo as compared toCompound No. 396443. As shown in Example 5, Compound No. 1263789,Compound No. 1287745, and Compound No. 1358996 achieved an ED₅₀ inspinal cord of 13.3, 8.8, and 7.4, respectively. In comparison, CompoundNo. 396443 achieved an ED₅₀ in spinal cord of 22.0. Therefore, each ofCompound No. 1263789, Compound No. 1287745, and Compound No. 1358996 aremore potent than Compound No. 396443 in this assay.

For example, Compound No. 1263789, Compound No. 1287717, Compound No.1287745, and Compound No. 1358996 each demonstrated improved 3 hour FOBscores as compared to Compound No. 396443, Compound No. 387954, andCompound No. 443305. As shown in Example 6, at 700 μg, Compound No.1263789, Compound No. 1287717, Compound No. 1287745, and Compound No.1358996 achieved 3 hour FOB scores of 0, 3.25, 1, and 0, respectively.In comparison, at half the dose (350 μg) Compound No. 396443 achieved a3 hour FOB score of 4.0; and at the same dose (700 μg) Compound No.387954 and Compound No. 443305 achieved a 3 hour FOB score of 4.0 and4.75, respectively. Therefore, each of Compound No. 1263789, CompoundNo. 1287717, Compound No. 1287745, and Compound No. 1358996 are moretolerable than Compound No. 396443, Compound No. 387954, and CompoundNo. 443305 in this assay.

For example, Compound No. 1263789, Compound No. 1287717, Compound No.1287745, and Compound No. 1358996 each demonstrated improved long-termtolerability as compared to Compound No. 396442 and Compound No. 819735.As shown in Example 7, Compound No. 1263789, Compound No. 1287717,Compound No. 1287745, and Compound No. 1358996 demonstrated no adverseevents, no Purkinje cell loss, and cortex GFAP mRNA less than 2-fold ofcontrol. In comparison, 396442 and 819735 each demonstrated adverseevents, Purkinje cell loss, and cortex GFAP mRNA greater than 2-fold ofcontrol in certain treated animals Therefore, each of Compound No.1263789, Compound No. 1287717, Compound No. 1287745, and Compound No.1358996 are more tolerable than Compound No. 396442 and Compound No.819735 in this assay.

Nonlimiting Disclosure and Incorporation by Reference

Each of the literature and patent publications listed herein isincorporated by reference in its entirety. While certain compounds,compositions and methods described herein have been described withspecificity in accordance with certain embodiments, the followingexamples serve only to illustrate the compounds described herein and arenot intended to limit the same. Each of the references, GenBankaccession numbers, and the like recited in the present application isincorporated herein by reference in its entirety.

Although the sequence listing accompanying this filing identifies eachsequence as either “RNA” or “DNA” as required, in reality, thosesequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides is,in certain instances, arbitrary. For example, an oligonucleotidecomprising a nucleoside comprising a 2′-OH sugar moiety and a thyminebase could be described as a DNA having a modified sugar moiety (2′-OHin place of one 2′-H of DNA) or as an RNA having a modified base(thymine (methylated uracil) in place of a uracil of RNA). Accordingly,nucleic acid sequences provided herein, including, but not limited tothose in the sequence listing, are intended to encompass nucleic acidscontaining any combination of natural or modified RNA and/or DNA,including, but not limited to such nucleic acids having modifiednucleobases. By way of further example and without limitation, anoligomeric compound having the nucleobase sequence “ATCGATCG”encompasses any oligomeric compounds having such nucleobase sequence,whether modified or unmodified, including, but not limited to, suchcompounds comprising RNA bases, such as those having sequence “AUCGAUCG”and those having some DNA bases and some RNA bases such as “AUCGATCG”and oligomeric compounds having other modified nucleobases, such as“ATmCGAUCG,” wherein ^(m)C indicates a cytosine base comprising a methylgroup at the 5-position.

Certain compounds described herein (e.g., modified oligonucleotides)have one or more asymmetric center and thus give rise to enantiomers,diastereomers, and other stereoisomeric configurations that may bedefined, in terms of absolute stereochemistry, as (R) or (S), as a orsuch as for sugar anomers, or as (D) or (L), such as for amino acids,etc. Compounds provided herein that are drawn or described as havingcertain stereoisomeric configurations include only the indicatedcompounds. Compounds provided herein that are drawn or described withundefined stereochemistry include all such possible isomers, includingtheir stereorandom and optically pure forms, unless specified otherwise.Likewise, all cis- and trans-isomers and tautomeric forms of thecompounds herein are also included unless otherwise indicated.Oligomeric compounds described herein include chirally pure or enrichedmixtures as well as racemic mixtures. For example, oligomeric compoundshaving a plurality of phosphorothioate internucleoside linkages includesuch compounds in which chirality of the phosphorothioateinternucleoside linkages is controlled or is random. Unless otherwiseindicated, compounds described herein are intended to includecorresponding salt forms.

The compounds described herein include variations in which one or moreatoms are replaced with a non-radioactive isotope or radioactive isotopeof the indicated element. For example, compounds herein that comprisehydrogen atoms encompass all possible deuterium substitutions for eachof the¹H hydrogen atoms. Isotopic substitutions encompassed by thecompounds herein include but are not limited to: ²H or ³H in place of¹H, ¹³C or ¹⁴C in place of ¹²C, ¹⁵N in place of ¹⁴N, ¹⁷O or ¹⁸O in placeof ¹⁶O and ³³S, ³⁴S, ³⁵S, or ³⁶S in place of ³²S. In certainembodiments, non-radioactive isotopic substitutions may impart newproperties on the oligomeric compound that are beneficial for use as atherapeutic or research tool. In certain embodiments, radioactiveisotopic substitutions may make the compound suitable for research ordiagnostic purposes such as imaging.

EXAMPLES

The following examples illustrate certain embodiments of the presentdisclosure and are not limiting. Moreover, where specific embodimentsare provided, the inventors have contemplated generic application ofthose specific embodiments.

Example 1: Design of Modified Oligonucleotides Complementary to a HumanSMN2 Nucleic Acid

Modified oligonucleotides complementary to a human SMN2 nucleic acidwere designed and synthesized as indicated in the tables below.

The modified oligonucleotides in the tables below are 16, 17, 18, 19, or20 nucleosides in length, as specified. The modified oligonucleotidescomprise 2′-MOE sugar moieties, 2′-NMA sugar moieties, cEt sugarmoieties, 2′-OMe sugar moieties, and/or 2′β-D-deoxyribosyl sugarmoieties, as specified. Each internucleoside linkage throughout themodified oligonucleotides is either a phosphorothioate internucleosidelinkage or a phosphodiester internucleoside linkage, as specified.Cytosines are either non-methylated cytosines or 5-methyl cytosines, asspecified.

Each modified oligonucleotide listed in the tables below is 100%complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14truncated from nucleotides 19939708 to Ser. No. 19/967,777), unlessspecifically stated otherwise. Non-complementary nucleobases arespecified in the Nucleobase Sequence column in underlined, bold,italicized font. Each modified oligonucleotide listed in the tablesbelow targets an active site on the SMN2 transcript for inclusion ofexon 7. “Start site” indicates the 5′-most nucleoside to which themodified oligonucleotide is complementary in the target nucleic acidsequence. “Stop site” indicates the 3′-most nucleoside to which themodified oligonucleotide is complementary in the target nucleic acidsequence.

Table 2

The modified oligonucleotides in Table 2 below are 16, 17, 18, 19 or 20nucleosides in length. Each nucleoside comprises a 2′-MOE sugar moiety.The sugar motif for each modified oligonucleotide is provided in theSugar Motif column, wherein each ‘e’ represents a 2′-MOE sugar moiety.Each internucleoside linkage is either a phosphorothioateinternucleoside linkage or a phosphodiester internucleoside linkage. Theinternucleoside linkage motif for each modified oligonucleotide isprovided in the Internucleoside Linkage Motif column, wherein each ‘s’represents a phosphorothioate internucleoside linkage, and each ‘o’represents a phosphodiester internucleoside linkage. Each cytosine is a5-methyl cytosine.

Each modified oligonucleotide listed in Table 2 below is 100%complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14truncated from nucleotides 19939708 to Ser. No. 19/967,777), unlessspecifically stated otherwise. Non-complementary nucleobases arespecified in the Nucleobase Sequence column in underlined, bold,italicized font. “Start site” indicates the 5′-most nucleoside to whichthe modified oligonucleotide is complementary in the target nucleic acidsequence. “Stop site” indicates the 3′-most nucleoside to which themodified oligonucleotide is complementary in the target nucleic acidsequence.

TABLE 22′-MOE modified oligonucleotides with PS or mixed PS/PO internucleoside linkagesSEQ SEQ ID ID No: 1 No: 1 SEQ Compound Nucleobase Sequence Sugar MotifInternucleoside Start Stop ID Number (5′ to 3′) (5′ to 3′)Linkage Motif (5′ to 3′) Site Site No. 1287063 ACTTTCATAATGCTGGCAGeeeeeeeeeeeeeeeeeee ssssssssssssssssss 27059 27077 24 1287048CACTTTCATAATGCTGGCAG eeeeeeeeeeeeeeeeeeee sssssssssssssssssss 2705927078 25 1287064 CACTTTCATAATGCTGGCA eeeeeeeeeeeeeeeeeeessssssssssssssssss 27060 27078 26 1287049 TCACTTTCATAATGCTGGCAeeeeeeeeeeeeeeeeeeee sssssssssssssssssss 27060 27079 27 1210340CTTTCATAATGCTGGC eeeeeeeeeeeeeeee sssssssssssssss 27061 27076 28 1212868CTTTCATAATGCTGGC eeeeeeeeeeeeeeee sssssssssooooss 27061 27076 28 1212867CTTTCATAATGCTGGC eeeeeeeeeeeeeeee ssssssssoooosss 27061 27076 28 1212863CTTTCATAATGCTGGC eeeeeeeeeeeeeeee ssssssooossssss 27061 27076 28 1212866CTTTCATAATGCTGGC eeeeeeeeeeeeeeee ssssssoooosssss 27061 27076 28 1212861CTTTCATAATGCTGGC eeeeeeeeeeeeeeee sssssooooosssss 27061 27076 28 1212860CTTTCATAATGCTGGC eeeeeeeeeeeeeeee sssssoooooossss 27061 27076 28 1212865CTTTCATAATGCTGGC eeeeeeeeeeeeeeee ssssoooosssssss 27061 27076 28 1212859CTTTCATAATGCTGGC eeeeeeeeeeeeeeee ssssooooooossss 27061 27076 28 1212851CTTTCATAATGCTGGC eeeeeeeeeeeeeeee sssosssosssosss 27061 27076 28 1212850CTTTCATAATGCTGGC eeeeeeeeeeeeeeee ssosssssssssoss 27061 27076 28 1212852CTTTCATAATGCTGGC eeeeeeeeeeeeeeee ssossossossosss 27061 27076 28 1212853CTTTCATAATGCTGGC eeeeeeeeeeeeeeee ssossossosososs 27061 27076 28 1212854CTTTCATAATGCTGGC eeeeeeeeeeeeeeee ssososososososs 27061 27076 28 1212864CTTTCATAATGCTGGC eeeeeeeeeeeeeeee ssoooosssssssss 27061 27076 28 1212855CTTTCATAATGCTGGC eeeeeeeeeeeeeeee soossssssssooss 27061 27076 28 1212856CTTTCATAATGCTGGC eeeeeeeeeeeeeeee sooosssssssooss 27061 27076 28 1212857CTTTCATAATGCTGGC eeeeeeeeeeeeeeee sooossssssoooss 27061 27076 28 1212858CTTTCATAATGCTGGC eeeeeeeeeeeeeeee soooosssssoooss 27061 27076 28 1210339ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssssssssssssssss 27061 27077 291212849 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssssssssssooooss 27061 2707729 1212848 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssssssssoooossss 2706127077 29 1212845 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee sssssssooossssss27061 27077 29 1212844 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeessssssoooossssss 27061 27077 29 1212843 ACTTTCATAATGCTGGCeeeeeeeeeeeeeeeee ssssssooooosssss 27061 27077 29 1212842ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee sssssoooooosssss 27061 27077 291212841 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee sssssooooooossss 27061 2707729 1212847 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssssoooossssssss 2706127077 29 1212832 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssossssssssssoss27061 27077 29 1212833 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeessosssssossssoss 27061 27077 29 1212834 ACTTTCATAATGCTGGCeeeeeeeeeeeeeeeee ssosssosssossoss 27061 27077 29 1212835ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssossossossososs 27061 27077 291212836 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssososososososss 27061 2707729 1212846 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssoooossssssssss 2706127077 29 1212837 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee soosssssssssooss27061 27077 29 1212838 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeesooossssssssooss 27061 27077 29 1212839 ACTTTCATAATGCTGGCeeeeeeeeeeeeeeeee sooosssssssoooss 27061 27077 29 1212840ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee soooossssssoooss 27061 27077 291263814 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssossssssssssssss 2706127078 21 1263816 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssossssssssssss27061 27078 21 1263818 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeessssssossssssssss 27061 27078 21 1263820 CACTTTCATAATGCTGGCeeeeeeeeeeeeeeeeee ssssssssossssssss 27061 27078 21 1263822CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssssssossssss 27061 27078 211263824 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssssssssossss 2706127078 21 1263826 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssssssssssoss27061 27078 21 1210342 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeessosssssssssssoss 27061 27078 21 1263778 CACTTTCATAATGCTGGCeeeeeeeeeeeeeeeeee sossssssssssssoss 27061 27078 21 1263781CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sosssssssssosssss 27061 27078 211263783 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sosssssssosssssss 2706127078 21 1263785 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sosssssosssssssss27061 27078 21 1263787 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeesosssosssssssssss 27061 27078 21 1263789 CACTTTCATAATGCTGGCeeeeeeeeeeeeeeeeee sososssssssssssss 27061 27078 21 1263791CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssosssssssssoss 27061 27078 211263793 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssosssssssoss 2706127078 21 1263795 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssssosssssoss27061 27078 21 1263797 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeessssssssssosssoss 27061 27078 21 1263799 CACTTTCATAATGCTGGCeeeeeeeeeeeeeeeeee ssssssssssssososs 27061 27078 21 1263800CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee soossssssssssssss 27061 27078 211263802 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sssoossssssssssss 2706127078 21 1263804 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sssssoossssssssss27061 27078 21 1263806 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeesssssssoossssssss 27061 27078 21 1263808 CACTTTCATAATGCTGGCeeeeeeeeeeeeeeeeee sssssssssoossssss 27061 27078 21 1263810CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sssssssssssoossss 27061 27078 211263812 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sssssssssssssooss 2706127078 21 1210343 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssosssssosssssoss27061 27078 21 1212825 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeesssssssooosssssss 27061 27078 21 1212817 CACTTTCATAATGCTGGCeeeeeeeeeeeeeeeeee soossssssssssooss 27061 27078 21 1212824CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sssssssoooossssss 27061 27078 211212826 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssoooosssssssssss 2706127078 21 1212827 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssoooosssssssss27061 27078 21 1212828 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeessssssoooosssssss 27061 27078 21 1212829 CACTTTCATAATGCTGGCeeeeeeeeeeeeeeeeee ssssssssoooosssss 27061 27078 21 1212830CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssssssoooosss 27061 27078 211212831 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sssssssssssooooss 2706127078 21 1212818 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sooosssssssssooss27061 27078 21 1212823 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeessssssooooossssss 27061 27078 21 1212819 CACTTTCATAATGCTGGCeeeeeeeeeeeeeeeeee sooossssssssoooss 27061 27078 21 1212822CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssoooooosssss 27061 27078 211212820 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee soooosssssssoooss 2706127078 21 1212821 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sssssooooooosssss27061 27078 21 1287065 TCACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeeessssssssssssssssss 27061 27079 30 1210341 ACTTTCATAATGCTGGeeeeeeeeeeeeeeee sssssssssssssss 27062 27077 31 524403 CACTTTCATAATGCTGGeeeeeeeeeeeeeeeee ssssssssssssssss 27062 27078 32 1287121TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee ssssssssssssssoss 27062 27079 231287120 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sssssssssssssosss 2706227079 23 1287113 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sssssssssssssooss27062 27079 23 1287110 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeessssssssssssososs 27062 27079 23 1287119 TCACTTTCATAATGCTGGeeeeeeeeeeeeeeeeee sssssssssssosssss 27062 27079 23 1364782TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sssssssssssososss 27062 27079 231364777 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee ssssssssssossosss 2706227079 23 1287118 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sssssssssosssssss27062 27079 23 1364783 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeesssssssssosssosss 27062 27079 23 1287109 TCACTTTCATAATGCTGGeeeeeeeeeeeeeeeeee ssssssssosssssoss 27062 27079 23 1364784TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee ssssssssossssosss 27062 27079 231287117 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sssssssosssssssss 2706227079 23 1287112 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sssssssoossssssss27062 27079 23 1287116 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeesssssosssssssssss 27062 27079 23 1287115 TCACTTTCATAATGCTGGeeeeeeeeeeeeeeeeee sssosssssssssssss 27062 27079 23 1287114TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sosssssssssssssss 27062 27079 231287106 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sossssssssssssoss 2706227079 23 1287107 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sossssssossssssss27062 27079 23 1287108 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeesososssssssssssss 27062 27079 23 1287111 TCACTTTCATAATGCTGGeeeeeeeeeeeeeeeeee soossssssssssssss 27062 27079 23 1287066TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee ssssssssssssssssss 27062 2708033 1287074 TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee sssssssssssssssoss27062 27080 33 1287071 TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeesssssssssssssososs 27062 27080 33 1287073 TTCACTTTCATAATGCTGGeeeeeeeeeeeeeeeeeee ssssssssosssssssss 27062 27080 33 1287070TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee ssssssssossssssoss 27062 2708033 1287072 TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee sossssssssssssssss27062 27080 33 1287067 TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeesosssssssssssssoss 27062 27080 33 1287068 TTCACTTTCATAATGCTGGeeeeeeeeeeeeeeeeeee sossssssosssssssss 27062 27080 33 1287069TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee sosossssssssssssss 27062 2708033 1287060 ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeeessssssssssssssssoss  27062 27081 20 1287057 ATTCACTTTCATAATGCTGGeeeeeeeeeeeeeeeeeeee sssssssssssssssooss 27062 27081 20 1287054ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeee ssssssssssssssososs 2706227081 20 1287059 ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeeesssssssssosssssssss 27062 27081 20 1287053 ATTCACTTTCATAATGCTGGeeeeeeeeeeeeeeeeeeee sssssssssossssssoss 27062 27081 20 1287056ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeee ssssssssoosssssssss 2706227081 20 1287058 ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeeesosssssssssssssssss 27062 27081 20 1287050 ATTCACTTTCATAATGCTGGeeeeeeeeeeeeeeeeeeee sossssssssssssssoss 27062 27081 20 1287051ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeee sosssssssosssssssss 2706227081 20 1287052 ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeeesososssssssssssssss 27062 27081 20 1287055 ATTCACTTTCATAATGCTGGeeeeeeeeeeeeeeeeeeee soossssssssssssssss 27062 27081 20 1287075ATTCACTTTCATAATGCTG eeeeeeeeeeeeeeeeeee ssssssssssssssssss 27063 2708134 1287062 AGATTCACTTTCATAATGCT eeeeeeeeeeeeeeeeeeee sssssssssssssssssss27064 27083 35 1287061 GATTCACTTTCATAATGCTG eeeeeeeeeeeeeeeeeeeesssssssssssssssssss 27063 27082 49 1287076 GATTCACTTTCATAATGCTeeeeeeeeeeeeeeeeeee ssssssssssssssssss 27064 27082 50 1287701TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeee ssssssssssssssssss 27062 27079 36 1287702TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeee ssssssssssssssssss 27062 27079 37

Table 3

The modified oligonucleotides in Table 3 below are 16, 17, 18, 19 or 20nucleosides in length. Each nucleoside comprises a 2′-NMA sugar moiety.The sugar motif for each modified oligonucleotide is provided in theSugar Motif column, wherein each ‘n’ represents a 2′-NMA sugar moiety.Each internucleoside linkage is either a phosphorothioateinternucleoside linkage or a phosphodiester internucleoside linkage. Theinternucleoside linkage motif for each modified oligonucleotide isprovided in the Internucleoside Linkage Motif column, wherein each ‘s’represents a phosphorothioate internucleoside linkage, and each ‘o’represents a phosphodiester internucleoside linkage. Each cytosine is a5-methyl cytosine.

Each modified oligonucleotide listed in Table 3 below is 100%complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14truncated from nucleotides 19939708 to Ser. No. 19/967,777). “Startsite” indicates the 5′-most nucleoside to which the modifiedoligonucleotide is complementary in the target nucleic acid sequence.“Stop site” indicates the 3′-most nucleoside to which the modifiedoligonucleotide is complementary in the target nucleic acid sequence.

TABLE 32′-NMA modified oligonucleotides with PS or mixed PS/PO internucleoside linkagesSEQ SEQ ID ID Internucleoside No: 1 No: 1 SEQ CompoundNucleobase Sequence Sugar Motif Linkage Motif Start Stop ID Number(5′ to 3′) (5′ to 3′) (5′ to 3′) Site Site No. 1287127CACTTTCATAATGCTGGCA nnnnnnnnnnnnnnnnnnn ssssssssssssssssss 27060 2707826 1287122 TCACTTTCATAATGCTGGCA nnnnnnnnnnnnnnnnnnnn sssssssssssssssssss27060 27079 27 1212871 CTTTCATAATGCTGGC nnnnnnnnnnnnnnnn sssssssssssssss27061 27076 28 1212869 ACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnssssssssssssssss 27061 27077 29 1358996 CACTTTCATAATGCTGGCnnnnnnnnnnnnnnnnnn sososssssssssssss 27061 27078 21 1212873CACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnn ssosssssssssssoss 27061 27078 211212874 CACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnn ssosssssosssssoss 2706127078 21 1212875 CACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnn ssosssosssosssoss27061 27078 21 1212879 CACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnnsoossssssssssooss 27061 27078 21 1212880 CACTTTCATAATGCTGGCnnnnnnnnnnnnnnnnnn sooosssssssssooss 27061 27078 21 1212881CACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnn sooossssssssoooss 27061 27078 211212885 CACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnn ssssssooooossssss 2706127078 21 1212887 CACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnn sssssssooosssssss27061 27078 21 1287128 TCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnnnssssssssssssssssss 27061 27079 30 1212870 CACTTTCATAATGCTGGnnnnnnnnnnnnnnnnn ssssssssssssssss 27062 27078 32 1287132TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnn sossssssssssssoss 27062 27079 231287133 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnn ssssssssossssssss 2706227079 23 1332246 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnn ssssssssosssssoss27062 27079 23 1332265 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnssssssssssssososs 27062 27079 23 1364778 TCACTTTCATAATGCTGGnnnnnnnnnnnnnnnnnn sssssssssssososss 27062 27079 23 1364779TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnn ssssssssssossosss 27062 27079 231364780 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnn sssssssssosssosss 2706227079 23 1364781 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnn ssssssssossssosss27062 27079 23 1287129 TTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnnssssssssssssssssss 27062 27080 33 1287130 TTCACTTTCATAATGCTGGnnnnnnnnnnnnnnnnnnn sosssssssssssssoss 27062 27080 33 1287131TTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn ssssssssosssssssss 27062 2708033 1332263 TTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn ssssssssossssssoss27062 27080 33 1332264 TTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnnsssssssssssssssoss 27062 27080 33 1332266 TTCACTTTCATAATGCTGGnnnnnnnnnnnnnnnnnnn sssssssssssssososs 27062 27080 33 1332270TTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn ssssssssssssssooss 27062 2708033 1287124 ATTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnnn sssssssssssssssssss27062 27081 20 1287125 ATTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnnnsossssssssssssssoss 27062 27081 20 1287126 ATTCACTTTCATAATGCTGGnnnnnnnnnnnnnnnnnnnn sssssssssosssssssss 27062 27081 20 1332267ATTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnnn ssssssssssssssssoss 2706227081 20 1332268 ATTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnnnsssssssssssssssooss 27062 27081 20 1332269 ATTCACTTTCATAATGCTGGnnnnnnnnnnnnnnnnnnnn ssssssssssssssososs 27062 27081 20 1332271ATTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnnn sssssssssossssssoss 2706227081 20 1287123 TTCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnnnnsssssssssssssssssss 27061 27080 22

Table 4

The modified oligonucleotides in Table 4 below are 18 or 19 nucleosidesin length. Each nucleoside comprises either a 2′-MOE sugar moiety or a2′-NMA sugar moiety. The sugar motif for each modified oligonucleotideis provided in the Sugar Motif column, wherein each ‘e’ represents a2′-MOE sugar moiety, and each ‘n’ represents a 2′-NMA sugar moiety. Eachinternucleoside linkage is a phosphorothioate internucleoside linkage.The internucleoside linkage motif for each modified oligonucleotide isprovided in the Internucleoside Linkage Motif column, wherein each ‘s’represents a phosphorothioate internucleoside linkage. Each cytosine isa 5-methyl cytosine.

Each modified oligonucleotide listed in Table 4 below is 100%complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14truncated from nucleotides 19939708 to Ser. No. 19/967,777), unlessspecifically stated otherwise. Non-complementary nucleobases arespecified in the Nucleobase Sequence column in underlined, bold,italicized font. “Start site” indicates the 5′-most nucleoside to whichthe modified oligonucleotide is complementary in the target nucleic acidsequence. “Stop site” indicates the 3′-most nucleoside to which themodified oligonucleotide is complementary in the target nucleic acidsequence.

TABLE 4Mixed 2′-MOE/2′-NMA modified oligonucleotides with PS internucleoside linkagesSEQ SEQ ID ID Internucleoside No: 1 No: 1 SEQ CompoundNucleobase Sequence Sugar Motif Linkage Motif Start Stop ID Number(5′ to 3′) (5′ to 3′) (5′ to 3′) Site Site No. 1212931CACTTTCATAATGCTGGC nennnnneneennnnnnn sssssssssssssssss 27061 27078 211212936 CACTTTCATAATGCTGGC nnnnnnnnnmmnenneen sssssssssssssssss 2706127078 21 1212941 CACTTTCATAATGCTGGC nennnnneneenenneen sssssssssssssssss27061 27078 21 1287728 TCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnnessssssssssssssssss 27061 27079 30 1287729 TCACTTTCATAATGCTGG

nnnnnnnnnnnnnnnnnne ssssssssssssssssss 27062 27079 36 1287730TCACTTTCATAATGCTGG

nnnnnnnnnnnnnnnnnne ssssssssssssssssss 27062 27079 37

Table 5

The modified oligonucleotides in Table 5 below are 16, 17, or 18nucleosides in length. Each nucleoside comprises either a 2′-MOE sugarmoiety or a cEt sugar moiety. The sugar motif for each modifiedoligonucleotide is provided in the Sugar Motif column, wherein each ‘e’represents a 2′-MOE sugar moiety, and each ‘k’ represents a cEt sugarmoiety. Each internucleoside linkage is a phosphorothioateinternucleoside linkage. The internucleoside linkage motif for eachmodified oligonucleotide is provided in the Internucleoside LinkageMotif column, wherein each ‘s’ represents a phosphorothioateinternucleoside linkage. Each cytosine is a 5-methyl cytosine.

Each modified oligonucleotide listed in Table 5 below is 100%complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14truncated from nucleotides 19939708 to Ser. No. 19/967,777). “Startsite” indicates the 5′-most nucleoside to which the modifiedoligonucleotide is complementary in the target nucleic acid sequence.“Stop site” indicates the 3′-most nucleoside to which the modifiedoligonucleotide is complementary in the target nucleic acid sequence.

TABLE 5Mixed 2′-MOE/cEt modified oligonucleotides with PS internucleoside linkagesSEQ SEQ ID ID Internucleoside No: 1 No: 1 SEQ CompoundNucleobase Sequence Sugar Motif Linkage Motif Start Stop ID Number(5′ to 3′) (5′ to 3′) (5′ to 3′) Site Site No. 1212961CACTTTCATAATGCTGGC keekeekeekeekeeeek sssssssssssssssss 27061 27078 211212962 CACTTTCATAATGCTGGC keeekeeekeeekeeeek sssssssssssssssss 2706127078 21 1212963 CACTTTCATAATGCTGGC keeeeekeeeeekeeeek sssssssssssssssss27061 27078 21 1212964 CACTTTCATAATGCTGGC keeeeeeekeeeeeeeeksssssssssssssssss 27061 27078 21 1212965 CACTTTCATAATGCTGGCkeeeeeeeeeeeeeeeek sssssssssssssssss 27061 27078 21 1212966CACTTTCATAATGCTGGC eeekeekeekeekeekek sssssssssssssssss 27061 27078 211212967 CACTTTCATAATGCTGGC eeekeekeekeekeekee sssssssssssssssss 2706127078 21 1212968 CACTTTCATAATGCTGGC eeeeeekeekeekeekee sssssssssssssssss27061 27078 21 1212969 CACTTTCATAATGCTGGC eeeeeekeekeekeeeeesssssssssssssssss 27061 27078 21 1212970 CACTTTCATAATGCTGGCeeeeeekeeeeekeeeee sssssssssssssssss 27061 27078 21 1212971CACTTTCATAATGCTGGC keekeekeekeeeeeeee sssssssssssssssss 27061 27078 211212972 CACTTTCATAATGCTGGC eeeeeeeekeekeekeek sssssssssssssssss 2706127078 21 1212973 CACTTTCATAATGCTGGC keekeekeeeeeeeeeee sssssssssssssssss27061 27078 21 1212974 CACTTTCATAATGCTGGC eeeeeeeeeeekeekeeksssssssssssssssss 27061 27078 21 1212975 CACTTTCATAATGCTGGCkeekeeeeeeeeeeeeee sssssssssssssssss 27061 27078 21 1212976CACTTTCATAATGCTGGC eeeeeeeeeeeeeekeek sssssssssssssssss 27061 27078 211212977 ACTTTCATAATGCTGGC keekeekeekeekeeek ssssssssssssssss 27061 2707729 1212978 ACTTTCATAATGCTGGC keeekeeekeeekeeek ssssssssssssssss 2706127077 29 1212979 ACTTTCATAATGCTGGC keeeekeeeeekeeeek ssssssssssssssss27061 27077 29 1212980 ACTTTCATAATGCTGGC keeeeeeekeeeeeeekssssssssssssssss 27061 27077 29 1212981 ACTTTCATAATGCTGGCkeeeeeeeeeeeeeeek ssssssssssssssss 27061 27077 29 1212982ACTTTCATAATGCTGGC eekeekeekeekeekek ssssssssssssssss 27061 27077 291212983 ACTTTCATAATGCTGGC eekeekeekeekeekee ssssssssssssssss 27061 2707729 1212984 ACTTTCATAATGCTGGC eeeeekeekeekeekee ssssssssssssssss 2706127077 29 1212985 ACTTTCATAATGCTGGC eeeeekeekeekeeeee ssssssssssssssss27061 27077 29 1212986 ACTTTCATAATGCTGGC eeeeekeeeeekeeeeessssssssssssssss 27061 27077 29 1212987 ACTTTCATAATGCTGGCkeekeekeekeeeeeee ssssssssssssssss 27061 27077 29 1212988ACTTTCATAATGCTGGC eeeeeeekeekeekeek ssssssssssssssss 27061 27077 291212989 ACTTTCATAATGCTGGC keekeekeeeeeeeeee ssssssssssssssss 27061 2707729 1212990 ACTTTCATAATGCTGGC eeeeeeeeeekeekeek ssssssssssssssss 2706127077 29 1212991 ACTTTCATAATGCTGGC keekeeeeeeeeeeeee ssssssssssssssss27061 27077 29 1212992 ACTTTCATAATGCTGGC eeeeeeeeeeeeekeekssssssssssssssss 27061 27077 29 1212993 CTTTCATAATGCTGGCkeekeekeekeekeek sssssssssssssss 27061 27076 28 1212994 CTTTCATAATGCTGGCkeeekeeekeeekeek sssssssssssssss 27061 27076 28 1212995 CTTTCATAATGCTGGCkeeeekeeeekeeeek sssssssssssssss 27061 27076 28 1212996 CTTTCATAATGCTGGCkeeeeeeekeeeeeek sssssssssssssss 27061 27076 28 1212997 CTTTCATAATGCTGGCkeeeeeeeeeeeeeek sssssssssssssss 27061 27076 28 1212998 CTTTCATAATGCTGGCkekeekeekeekeeke sssssssssssssss 27061 27076 28 1212999 CTTTCATAATGCTGGCeekeekeekeekeeke sssssssssssssss 27061 27076 28 1213000 CTTTCATAATGCTGGCeeeeekeekeekeeke sssssssssssssss 27061 27076 28 1213001 CTTTCATAATGCTGGCeeeeekeekeekeeee sssssssssssssss 27061 27076 28 1213002 CTTTCATAATGCTGGCeeeeekeeeeekeeee sssssssssssssss 27061 27076 28 1213003 CTTTCATAATGCTGGCkeekeekeekeeeeee sssssssssssssss 27061 27076 28 1213004 CTTTCATAATGCTGGCeeeeeekeekeekeek sssssssssssssss 27061 27076 28 1213005 CTTTCATAATGCTGGCkeekeekeeeeeeeee sssssssssssssss 27061 27076 28 1213006 CTTTCATAATGCTGGCeeeeeeeeekeekeek sssssssssssssss 27061 27076 28 1213007 CTTTCATAATGCTGGCkeekeeeeeeeeeeee sssssssssssssss 27061 27076 28 1213008 CTTTCATAATGCTGGCeeeeeeeeeeeekeek sssssssssssssss 27061 27076 28

Table 6

The modified oligonucleotides in Table 6 below are 19 or 20 nucleosidesin length. Each nucleoside comprises a 2′-MOE sugar moiety, a 2′-NMAsugar moiety, a 2′-OMe sugar moiety, or a 2′β-D-deoxyribosyl sugarmoiety. The sugar motif for each modified oligonucleotide is provided inthe Sugar Motif column, wherein each ‘e’ represents a 2′-MOE sugarmoiety, each ‘n’ represents a 2′-NMA sugar moiety, each ‘y’ represents a2′-OMe sugar moiety, and each ‘d’ represents a 2′β-D-deoxyribosyl sugarmoiety. Each internucleoside linkage is either a phosphorothioateinternucleoside linkage or a phosphodiester internucleoside linkage. Theinternucleoside linkage motif for each modified oligonucleotide,provided in the Internucleoside Linkage Motif column, is (from 5′ to3′): ssssssssssssssssso; wherein each ‘s’ represents a phosphorothioateinternucleoside linkage, and each ‘o’ represents a phosphodiesterinternucleoside linkage. Cytosines are either non-methylated cytosinesor 5-methyl cytosines, wherein each lowercase ‘c’ in the NucleobaseSequence column represents a non-methylated cytosine, and each uppercase‘C’ in the Nucleobase Sequence column represents a 5-methyl cytosine.

Each nucleobase in the modified oligonucleotides listed in Table 6 belowis complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14truncated from nucleotides 19939708 to Ser. No. 19/967,777), unlessspecifically stated otherwise. Non-complementary nucleobases arespecified in the Nucleobase Sequence column in underlined, bold,italicized font. “Start site” indicates the 5′-most nucleoside to whichthe modified oligonucleotide is complementary in the target nucleic acidsequence. “Stop site” indicates the 3′-most nucleoside to which themodified oligonucleotide is complementary in the target nucleic acidsequence.

TABLE 6Modified oligonucleotides with mixed PS/PO internucleoside linkages SEQSEQ ID ID Internucleoside No: 1 No: 1 SEQ Compound Nucleobase SequenceSugar Motif Linkage Motif Start Stop ID Number (5′ to 3′) (5′ to 3′)(5′ to 3′) Site Site No. 1287707 TCACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeedssssssssssssssssso 27061 27079 30 1287708 TCACTTTCATAATGCTGGceeeeeeeeeeeeeeeeeed ssssssssssssssssso 27061 27079 30 1287709TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeed ssssssssssssssssso 27062 27079 36 1287710TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeed ssssssssssssssssso 27062 27079 37 1287711TCACTTTCATAATGCTGGc eeeeeeeeeeeeeeeeeey ssssssssssssssssso 27061 2707930 1287712 TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeey ssssssssssssssssso 27062 27079 38 1287713TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeey ssssssssssssssssso 27062 27079 37 1287731TCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnne ssssssssssssssssso 27061 2707930 1287732 TCACTTTCATAATGCTGGc nnnnnnnnnnnnnnnnnne ssssssssssssssssso27061 27079 30 1287733 TCACTTTCATAATGCTGG

nnnnnnnnnnnnnnnnnne ssssssssssssssssso 27062 27079 36 1287734TCACTTTCATAATGCTGG

nnnnnnnnnnnnnnnnnne ssssssssssssssssso 27062 27079 37 1287735TCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnnd ssssssssssssssssso 27061 2707930 1287736 TCACTTTCATAATGCTGGc nnnnnnnnnnnnnnnnnnd ssssssssssssssssso27061 27079 30 1287737 TCACTTTCATAATGCTGG

nnnnnnnnnnnnnnnnnnd ssssssssssssssssso 27062 27079 36 1287738TCACTTTCATAATGCTGG

nnnnnnnnnnnnnnnnnnd ssssssssssssssssso 27062 27079 37 1287739TCACTTTCATAATGCTGGc nnnnnnnnnnnnnnnnnny ssssssssssssssssso 27061 2707930 1287740 TCACTTTCATAATGCTGG

nnnnnnnnnnnnnnnnnny ssssssssssssssssso 27062 27079 38 1287741TCACTTTCATAATGCTGG

nnnnnnnnnnnnnnnnnny ssssssssssssssssso 27062 27079 37 1287705TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeee ssssssssssssssssso 27062 27079 36 1287706TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeee ssssssssssssssssso 27062 27079 37 1287704TCACTTTCATAATGCTGGc eeeeeeeeeeeeeeeeeee ssssssssssssssssso 27061 2707930 1287703 TCACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeee ssssssssssssssssso27061 27079 30

Table 7

The modified oligonucleotides in Table 7 below are 19 or 20 nucleosidesin length. Each nucleoside comprises a 2′-MOE sugar moiety, a 2′-NMAsugar moiety, or a 2′β-D-deoxyribosyl sugar moiety. The sugar motif foreach modified oligonucleotide is provided in the Sugar Motif column,wherein each ‘e’ represents a 2′-MOE sugar moiety, each ‘n’ represents a2′-NMA sugar moiety, and each ‘d’ represents a 2′β-D-deoxyribosyl sugarmoiety. Each internucleoside linkage is either a phosphorothioateinternucleoside linkage or a phosphodiester internucleoside linkage. Theinternucleoside linkage motif for each modified oligonucleotide,provided in the Internucleoside Linkage Motif column, is (from 5′ to3′): sssssssssssssssssoo; wherein each ‘s’ represents a phosphorothioateinternucleoside linkage, and each ‘o’ represents a phosphodiesterinternucleoside linkage. Each cytosine is a 5-methyl cytosine. Eachnucleobase in the modified oligonucleotide listed in Table 6 below iscomplementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14truncated from nucleotides 19939708 to Ser. No. 19/967,777), unlessspecifically stated otherwise. Non-complementary nucleobases arespecified in the Nucleobase Sequence column in underlined, bold,italicized font. “Start site” indicates the 5′-most nucleoside to whichthe modified oligonucleotide is complementary in the target nucleic acidsequence. “Stop site” indicates the 3′-most nucleoside to which themodified oligonucleotide is complementary in the target nucleic acidsequence.

TABLE 7Modified oligonucleotides with mixed PS/PO internucleoside linkages SEQSEQ ID ID Internucleoside No: 1 No: 1 SEQ Compound Nucleobase SequenceSugar Motif Linkage Motif Start Stop ID Number (5 to 3′) (5′ to 3′)(5′ to 3′) Site Site No. 1318749 TCACTTTCATAATGCTGG

A nnnnnnnnnnnnnnnnnndd sssssssssssssssssoo 27062 27079 39 1318750TCACTTTCATAATGCTGGCA nnnnnnnnnnnnnnnnnned sssssssssssssssssoo 2706027079 27 1318751 TCACTTTCATAATGCTGGCA nnnnnnnnnnnnnnnnnnddsssssssssssssssssoo 27060 27079 27 1318752 TCACTTTCATAATGCTGG

A nnnnnnnnnnnnnnnnnned sssssssssssssssssoo 27062 27079 39 1318753TCACTTTCATAATGCTGG

A nnnnnnnnnnnnnnnnnnde sssssssssssssssssoo 27062 27079 39 1318754TCACTTTCATAATGCTGGCA nnnnnnnnnnnnnnnnnnde sssssssssssssssssoo 2706027079 27 1318755 TCACTTTCATAATGCTGG

A nnnnnnnnnnnnnnnnnnee sssssssssssssssssoo 27062 27079 39 1318756TCACTTTCATAATGCTGGCA nnnnnnnnnnnnnnnnnnee sssssssssssssssssoo 2706027079 27 1318757 TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 27062 27079 40 1318758TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 27062 27079 41 1318759TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 27062 27079 42 1318760TCACTTTCATAATGCTGG

A eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 27062 27079 39 1318761TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 27062 27079 43 1318762TCACTTTCATAATGCTGG

A eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 27062 27079 44 1318763TCACTTTCATAATGCTGGC

eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 27061 27079 45 1318764TCACTTTCATAATGCTGG

A eeeeeeeeeeeeeeeeeeed sssssssssssssssssoo 27062 27079 39 1318765TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeede sssssssssssssssssoo 27062 27079 41 1318766TCACTTTCATAATGCTGGC

eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 27061 27079 46 1318767TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeede sssssssssssssssssoo 27062 27079 43 1318768TCACTTTCATAATGCTGGCA eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 2706027079 27 1318769 TCACTTTCATAATGCTGGCA eeeeeeeeeeeeeeeeeeedsssssssssssssssssoo 27060 27079 27 1318770 TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeede sssssssssssssssssoo 27062 27079 40 1318771TCACTTTCATAATGCTGG

A eeeeeeeeeeeeeeeeeede sssssssssssssssssoo 27062 27079 44 1318772TCACTTTCATAATGCTGG

A eeeeeeeeeeeeeeeeeede sssssssssssssssssoo 27062 27079 39 1318773TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeede sssssssssssssssssoo 27062 27079 42 1318774TCACTTTCATAATGCTGGC

eeeeeeeeeeeeeeeeeede sssssssssssssssssoo 27061 27079 45 1318775TCACTTTCATAATGCTGGC

eeeeeeeeeeeeeeeeeede sssssssssssssssssoo 27061 27079 46 1318776TCACTTTCATAATGCTGGCA eeeeeeeeeeeeeeeeeede sssssssssssssssssoo 2706027079 27 1333508 TCACTTTCATAATGCTGGC

nnnnnnnnnnnnnnnnnnee sssssssssssssssssoo  27061 27079 46 1318777TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo 27062 27079 41 1318778TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo 27062 27079 43 1318779TCACTTTCATAATGCTGG

A eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo 27062 27079 39 1318780TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo 27062 27079 42 1318781TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo 27062 27079 40 1318782TCACTTTCATAATGCTGGC

eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo 27061 27079 46 1318783TCACTTTCATAATGCTGG

A eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo 27062 27079 44 1318784TCACTTTCATAATGCTGGC

eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo 27061 27079 45 1318748TCACTTTCATAATGCTGGCA eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo  2706027079 27

Table 8

The modified oligonucleotides in Table 8 below are each 19 nucleosidesin length. Each nucleoside comprises a 2′-MOE sugar moiety, a 2′-NMAsugar moiety, or a 2′β-D-deoxyribosyl sugar moiety. The sugar motif foreach modified oligonucleotide is provided in the Sugar Motif column,wherein each ‘e’ represents a 2′-MOE sugar moiety, each represents a2′-NMA sugar moiety, and each represents a 2′β-D-deoxyribosyl sugarmoiety. Each internucleoside linkage is either a phosphorothioateinternucleoside linkage or a phosphodiester internucleoside linkage. Theinternucleoside linkage motif for each modified oligonucleotide,provided in the Internucleoside Linkage Motif column, is (from 5′ to3′): ssssssssssssososso; wherein each represents a phosphorothioateinternucleoside linkage, and each represents a phosphodiesterinternucleoside linkage. Each cytosine is a 5-methyl cytosine. Eachnucleobase in the modified oligonucleotide listed in Table 8 below iscomplementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14truncated from nucleotides 19939708 to Ser. No. 19/967,777), unlessspecifically stated otherwise. Non-complementary nucleobases arespecified in the Nucleobase Sequence column in underlined, bold,italicized font. “Start site” indicates the 5′-most nucleoside to whichthe modified oligonucleotide is complementary in the target nucleic acidsequence. “Stop site” indicates the 3′-most nucleoside to which themodified oligonucleotide is complementary in the target nucleic acidsequence.

TABLE 8Modified oligonucleotides with mixed PS/PO internucleoside linkages SEQSEQ ID ID Internucleoside No: 1 No: 1 SEQ Compound Nucleobase SequenceSugar Motif Linkage Motif Start Stop ID Number (5′ to 3′) (5′ to 3′)(5′ to 3′) Site Site No. 1332247 TCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnndssssssssssssososso 27061 27079 30 1332248 TCACTTTCATAATGCTGG

nnnnnnnnnnnnnnnnnnd ssssssssssssososso 27062 27079 37 1332249TCACTTTCATAATGCTGG

nnnnnnnnnnnnnnnnnne ssssssssssssososso 27062 27079 37 1332251TCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnne ssssssssssssososso 27061 2707930 1332255 TCACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeed ssssssssssssososso27061 27079 30 1332257 TCACTTTCATAATGCTGGA eeeeeeeeeeeeeeeeeedssssssssssssososso 27062 27079 37 1332256 TCACTTTCATAATGCTGGAeeeeeeeeeeeeeeeeeee ssssssssssssososso 27062 27079 37 1332258TCACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeee ssssssssssssososso 27061 2707930

Table 9

The modified oligonucleotides in Table 9 below are each 19 nucleosidesin length. Each nucleoside comprises a 2′-MOE sugar moiety, a 2′-NMAsugar moiety, or a 2′β-D-deoxyribosyl sugar moiety. The sugar motif foreach modified oligonucleotide is provided in the Sugar Motif column,wherein each ‘e’ represents a 2′-MOE sugar moiety, each ‘n’ represents a2′-NMA sugar moiety, and each ‘d’ represents a 2′β-D-deoxyribosyl sugarmoiety. Each internucleoside linkage is either a phosphorothioateinternucleoside linkage or a phosphodiester internucleoside linkage. Theinternucleoside linkage motif for each modified oligonucleotide,provided in the Internucleoside Linkage Motif column, is (from 5′ to3′): ssssssssssssssosso; wherein each ‘s’ represents a phosphorothioateinternucleoside linkage, and each ‘o’ represents a phosphodiesterinternucleoside linkage. Each cytosine is a 5-methyl cytosine.

Each nucleobase in the modified oligonucleotide listed in Table 9 belowis complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14truncated from nucleotides 19939708 to Ser. No. 19/967,777), unlessspecifically stated otherwise. Non-complementary nucleobases arespecified in the Nucleobase Sequence column in underlined, bold,italicized font. “Start site” indicates the 5′-most nucleoside to whichthe modified oligonucleotide is complementary in the target nucleic acidsequence. “Stop site” indicates the 3′-most nucleoside to which themodified oligonucleotide is complementary in the target nucleic acidsequence.

TABLE 9Modified oligonucleotides with mixed PS/PO internucleoside linkages SEQSEQ ID ID Internucleoside No: 1 No: 1 SEQ Compound Nucleobase SequenceSugar Motif Linkage Motif Start Stop ID Number (5′ to 3′) (5′ to 3′)(5′ to 3′) Site Site No. 1332250 TCACTTTCATAATGCTGG

nnnnnnnnnnnnnnnnnnd ssssssssssssssosso 27062 27079 37 1332252TCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnnd ssssssssssssssosso 27061 2707930 1332253 TCACTTTCATAATGCTGG

nnnnnnnnnnnnnnnnnne ssssssssssssssosso 27062 27079 37 1332254TCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnne ssssssssssssssosso 27061 2707930 1332259 TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeed ssssssssssssssosso 27062 27079 37 1332260TCACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeed ssssssssssssssosso 27061 2707930 1332261 TCACTTTCATAATGCTGG

eeeeeeeeeeeeeeeeeee ssssssssssssssosso 27062 27079 37 1332262TCACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeee ssssssssssssssosso 27061 2707930

Table 10

The modified oligonucleotides in Table 10 below are each 19 nucleosidesin length. Each nucleoside comprises a 2′-MOE sugar moiety or a 2′-NMAsugar moiety. The sugar motif for each modified oligonucleotide isprovided in the Sugar Motif column, wherein each ‘e’ represents a 2′-MOEsugar moiety, and each ‘n’ represents a 2′-NMA sugar moiety. Eachinternucleoside linkage is either a phosphorothioate internucleosidelinkage or a phosphodiester internucleoside linkage. The internucleosidelinkage motif for each modified oligonucleotide, provided in theInternucleoside Linkage Motif column, is (from 5′ to 3′):osssssssssssssssss; wherein each ‘s’ represents a phosphorothioateinternucleoside linkage, and each ‘o’ represents a phosphodiesterinternucleoside linkage. Each cytosine is a 5-methyl cytosine.

Each nucleobase in the modified oligonucleotide listed in Table 10 belowis complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14truncated from nucleotides 19939708 to Ser. No. 19/967,777), unlessspecifically stated otherwise. Non-complementary nucleobases arespecified in the Nucleobase Sequence column in underlined, bold,italicized font. “Start site” indicates the 5′-most nucleoside to whichthe modified oligonucleotide is complementary in the target nucleic acidsequence. “Stop site” indicates the 3′-most nucleoside to which themodified oligonucleotide is complementary in the target nucleic acidsequence.

TABLE 10Modified oligonucleotides with mixed PS/PO internucleoside linkages SEQSEQ ID ID Internucleoside No: 1 No: 1 SEQ Compound Nucleobase SequenceSugar Motif Linkage Motif Start Stop ID Number (5′ to 3′) (5′ to 3′)(5′ to 3′) Site Site No. 1287742

TCACTTTCATAATGCTGG ennnnnnnnnnnnnnnnnn osssssssssssssssss 27062 27079 471287743 TTCACTTTCATAATGCTGG ennnnnnnnnnnnnnnnnn osssssssssssssssss 2706227080 33 1287744

TCACTTTCATAATGCTGG ennnnnnnnnnnnnnnnnn osssssssssssssssss 27062 27079 481287714

TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee osssssssssssssssss 27062 27079 471287716

TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee osssssssssssssssss 27062 27079 481287715 TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee osssssssssssssssss 2706227080 33

Table 11

The modified oligonucleotides in Table 11 below are each 20 nucleosidesin length. Each nucleoside comprises a 2′-MOE sugar moiety or a 2′-NMAsugar moiety. The sugar motif for each modified oligonucleotide isprovided in the Sugar Motif column, wherein each ‘e’ represents a 2′-MOEsugar moiety, and each ‘n’ represents a 2′-NMA sugar moiety. Eachinternucleoside linkage is either a phosphorothioate internucleosidelinkage or a phosphodiester internucleoside linkage. The internucleosidelinkage motif for each modified oligonucleotide, provided in theInternucleoside Linkage Motif column, is (from 5′ to 3′):ossssssssssssssssso; wherein each ‘s’ represents a phosphorothioateinternucleoside linkage, and each ‘o’ represents a phosphodiesterinternucleoside linkage. Each cytosine is a 5-methyl cytosine.

Each modified oligonucleotide listed in Table 11 below is 100%complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14truncated from nucleotides 19939708 to Ser. No. 19/967,777). “Startsite” indicates the 5′-most nucleoside to which the modifiedoligonucleotide is complementary in the target nucleic acid sequence.“Stop site” indicates the 3′-most nucleoside to which the modifiedoligonucleotide is complementary in the target nucleic acid sequence.

TABLE 11Modified oligonucleotides with mixed PS/PO internucleoside linkages SEQSEQ Internucleoside ID No: ID No: SEQ Compound Nucleobase SequenceSugar Motif Linkage Motif 1 Start  1 Stop ID Number (5′ to 3′)(5′ to 3′) (5′ to 3′) Site Site No. 1287745 TTCACTTTCATAATGCTGGCennnnnnnnnnnnnnnnnne ossssssssssssssssso 27061 27080 22 1287717TTCACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeeee ossssssssssssssssso 2706127080 22

Example 2: Activity of Modified Oligonucleotides Complementary to HumanSMN2 in Transgenic Mice, Single Dose (35 μg)

Activity of selected modified oligonucleotides described above wastested in human SMN2 transgenic mice. Taiwan strain of SMA Type III micewere obtained from The Jackson Laboratory (Bar Harbor, Me.). These micelack mouse SMN and are homozygous for human SMN2 (mSMN−/−; hSMN2+/+;FVB.Cg-Tg(SMN2)2HungSMN1tm1Hung/J, stock number 005058; Bar Harbor,Me.), or are heterozygous for mouse SMN and heterozygous for humanSMN2(mSMN+/−; hSMN2+/−; FVB.Cg-Tg(SMN2)2HungSMN1tm1Hung/J) obtained bybreeding HOM/HOM (stock #00005058) to FVB/NJ (Stock #001800).

Treatment

Homozygous or heterozygous transgenic mice were divided into groups of 4mice each. Each mouse received a single ICV bolus of 35 μg of modifiedoligonucleotide. Comparator Compound Nos. 387954, 396442, and 396443were also tested in this assay. A group of 4 mice received PBS as anegative control.

RNA Analysis

Two weeks post treatment, mice were sacrificed and RNA was extractedfrom cortical brain tissue and spinal cord for real-time qPCR analysisof SMN2 RNA. Primer probe set hSMN2vd#4_LTS00216_MGB (forward sequence:GCTGATGCTTTGGGAAGTATGTTA (SEQ ID NO: 11); reverse sequenceCACCTTCCTTCTTTTTGATTTTGTC, designated herein as SEQ ID NO: 12; probesequence TACATGAGTGGCTATCATACT (SEQ ID NO: 13)) was used to determinethe amount of SMN2 RNA including exon 7 (exon 71. Primer probe sethSMN2_Sumner68_PPS50481 (forward sequence: CATGGTACATGAGTGGCTATCATACTG(SEQ ID NO: 14); reverse sequence: TGGTGTCATTTAGTGCTGCTCTATG (SEQ ID NO:15); probe sequence CCAGCATTTCCATATAATAGC (SEQ ID NO: 16) was used todetermine the amount of SMN2 RNA excluding exon 7 (exon 7⁻). Total SMN2RNA levels were measured using primer probe set hSMN2_LTS00935 (forwardsequence: CAGGAGGATTCCGTGCTGTT (SEQ ID NO: 17); reverse sequenceCAGTGCTGTATCATCCCAAATGTC, (SEQ ID NO: 18); probe sequence:ACAGGCCAGAGCGAT (SEQ ID NO: 19)).

Results are presented as fold change in RNA levels relative to PBScontrol, normalized to total SMN2 levels. Each of Tables 12-18represents a different experiment.

TABLE 12 Effect of modified oligonucleotides on human SMN2 RNA splicingin homozygous transgenic mice Compound Dose CORTEX SPINAL CORD No. (μg)exon 7⁺ exon 7⁻ exon 7⁺ exon 7⁻ PBS — 1 1 1 1 396442 35 3.3 0.3 3.4 0.3396443 35 3.0 0.5 2.3 0.5 524403 35 3.3 0.4 2.5 0.5 1210339 35 2.5 0.53.0 0.3 1210340 35 2.1 0.6 2.6 0.4 1210341 35 1.8 0.7 2.0 0.6 1210342 352.5 0.5 2.9 0.3 1210343 35 3.0 0.4 2.4 0.5 1212817 35 2.4 0.6 2.2 0.61212818 35 2.4 0.5 2.1 0.6 1212823 35 2.0 0.6 2.0 0.6 1212824 35 2.1 0.62.1 0.6 1212825 35 2.9 0.4 2.5 0.5 1212826 35 2.5 0.6 2.2 0.7 1212827 352.5 0.6 2.6 0.5 1212828 35 2.9 0.5 2.4 0.6 1212830 35 2.8 0.7 2.1 0.81212831 35 2.5 0.7 2.3 0.7 1212832 35 2.9 0.6 2.9 0.5 1212833 35 2.4 0.72.7 0.5 1212837 35 2.5 0.6 2.7 0.5 1212838 35 2.1 0.7 2.5 0.6 1212844 352.6 0.6 2.4 0.7 1212845 35 2.3 0.7 2.5 0.7 1212846 35 2.8 0.6 2.6 0.61212849 35 2.1 0.7 2.3 0.6 1212850 35 1.8 0.8 2.2 0.7 1212855 35 2.0 0.72.1 0.8

TABLE 13 Effect of modified oligonucleotides on human SMN2 RNA splicingin homozygous transgenic mice Compound Dose CORTEX SPINAL CORD No. (μg)exon 7⁺ exon 7⁻ exon 7⁺ exon 7⁻ PBS — 1.0 1.0 1.0 1.0 396443 35 2.7 0.31.9 0.5 1210342 35 2.4 0.5 2.5 0.4 1212961 35 1.8 0.7 1.7 0.6 1212962 352.0 0.6 1.9 0.5 1212963 35 2.3 0.5 2.5 0.3 1212966 35 1.6 0.8 2.0 0.51212967 35 1.9 0.6 1.9 0.4 1212971 35 1.6 0.5 2.0 0.4 1212972 35 1.8 0.62.2 0.5 1212977 35 2.1 0.5 2.2 0.4 1212978 35 2.1 0.6 2.2 0.4 1212979 352.1 0.5 2.6 0.3 1212982 35 2.0 0.7 1.8 0.6 1212983 35 1.9 0.6 1.7 0.51212984 35 1.9 0.6 1.9 0.5 1212987 35 2.4 0.4 2.5 0.4 1212988 35 1.8 0.71.8 0.5 1212995 35 2.5 0.5 2.5 0.4 1212998 35 1.8 0.6 1.8 0.7 1212999 352.0 0.6 2.0 0.5 1213003 35 1.9 0.7 2.3 0.5 1213004 35 1.8 0.7 2.3 0.6

TABLE 14 Effect of modified oligonucleotides on human SMN2 RNA splicingin homozygous transgenic mice Compound Dose CORTEX SPINAL CORD No. (μg)exon 7⁺ exon 7⁻ exon 7⁺ exon 7⁻ PBS — 1.0 1.0 1.0 1.0 396443 35 2.6 0.53.1 0.5 1212964 35 2.5 0.6 3.6 0.4 1212965 35 2.9 0.5 3.3 0.4 1212968 352.2 0.6 2.3 0.6 1212973 35 2.6 0.5 3.2 0.4 1212974 35 2.3 0.6 2.8 0.51212975 35 2.9 0.3 3.1 0.4 1212976 35 2.5 0.5 2.8 0.5 1212980 35 2.6 0.53.2 0.4 1212981 35 2.9 0.4 3.6 0.3 1212985 35 2.4 0.6 2.9 0.5 1212986 352.8 0.4 3.3 0.4 1212989 35 3.3 0.3 3.6 0.2 1212990 35 1.8 0.8 2.1 0.71212991 35 3.2 0.3 3.8 0.3 1212992 35 2.4 0.5 2.2 0.6 1212996 35 2.2 0.63.2 0.5 1212997 35 2.9 0.4 3.9 0.4 1213001 35 2.1 0.5 2.8 0.6 1213002 352.0 0.6 2.9 0.6 1213005 35 2.8 0.5 3.2 0.3 1213006 35 1.9 0.9 2.0 0.81213007 35 3.3 0.2 2.9 0.5 1213008 35 2.3 0.7 2.2 0.7

TABLE 15 Effect of modified oligonucleotides on human SMN2 RNA splicingin heterozygous transgenic mice Compound Dose CORTEX SPINAL CORD No.(μg) exon 7⁺ exon 7⁻ exon 7⁺ exon 7⁻ PBS — 1.0 1.0 1.0 1.0 387954 35 2.30.6 2.2 0.5 396443 35 2.5 0.5 2.4 0.5 1287048 35 2.2 0.5 2.2 0.5 128704935 2.3 0.6 2.5 0.4 1287061 35 2.4 0.5 2.2 0.4 1287062 35 3.0 0.3 2.3 0.41287050 35 2.8 0.5 2.3 0.4 1287054 35 2.2 0.5 2.3 0.4 1287063 35 1.8 0.71.7 0.6 1287064 35 2.6 0.3 2.4 0.4 1287065 35 2.5 0.4 2.3 0.4 1287066 352.2 0.5 2 0.5 1287075 35 2.3 0.6 1.8 0.7 1287076 35 2.6 0.4 1.9 0.61287067 35 2.7 0.4 1.9 0.6 1287070 35 2.5 0.5 1.8 0.7 1287071 35 2.6 0.41.8 0.7 1287074 35 2.6 0.5 2 0.6 1287109 35 2.7 0.6 2.4 0.5 1287110 352.6 0.5 2.3 0.5 1287701 35 2.6 0.6 2.8 0.3 1287702 35 3 0.5 2.8 0.41287703 35 2.3 0.6 2.4 0.4 1287704 35 2.7 0.5 2.3 0.4 1287717 35 3.3 0.32.4 0.6

TABLE 16 Effect of modified oligonucleotides on human SMN2 RNA splicingin heterozygous transgenic mice Compound Dose CORTEX SPINAL CORD No.(μg) exon 7⁺ exon 7⁻ exon 7⁺ exon 7⁻ PBS — 1 1 1 1 396442 35 2.5 0.6 3.20.3 396443 35 3 0.5 2.8 0.5 1263783 35 3 0.3 2.6 0.5 1263785 35 3.1 0.42.9 0.5 1263787 35 2.4 0.6 2.9 0.4 1263789 35 3.8 0.2 2.6 0.5 1263800 353.6 0.2 2.6 0.5 1263802 35 3.4 0.3 2.9 0.4 1263806 35 3.5 0.2 2.7 0.51263808 35 3.2 0.4 2.7 0.5 1263810 35 2.8 0.5 2.4 0.5

TABLE 17 Effect of modified oligonucleotides on human SMN2 RNA splicingin heterozygous transgenic mice Compound Dose CORTEX SPINAL CORD No.(μg) exon 7⁺ exon 7⁻ exon 7⁺ exon 7⁻ PBS — 1 1 1 1 396443 35 2.5 0.6 2.90.4 1364784 35 2.3 0.7 2.8 0.5 1364783 35 2.9 0.5 2.4 0.5 1364777 35 2.70.6 2.3 0.5 1364782 35 2.7 0.6 2.6 0.5

TABLE 18 Effect of modified oligonucleotides on human SMN2 RNA splicingin heterozygous transgenic mice Compound Dose CORTEX SPINAL CORD No.(μg) exon 7⁺ exon 7⁻ exon 7⁺ exon 7⁻ PBS — 1 1 1 1 396443 35 2 0.7 2.70.5 1318748 35 2.1 0.7 2.5 0.6 1318782 35 2.2 0.8 2.5 0.6 1332262 35 3.30.4 2.9 0.5 1332258 35 2.4 0.7 2.3 0.6

Example 3: Activity of Modified Oligonucleotides Complementary to HumanSMN2 in Transgenic Mice, Single Dose (15 μg)

Activity of selected modified oligonucleotides described above wastested in human SMN2 transgenic mice essentially as described above inExample 2. Comparator Compound Nos. 396443 and 819735 were also testedin this assay. The transgenic mice were divided into groups of 4 miceeach. Each mouse received a single ICV bolus of 15 μg of modifiedoligonucleotide. A group of 4 mice received PBS as a negative control.Two weeks post treatment, mice were sacrificed and RNA was extractedfrom cortical brain tissue and spinal cord for real-time qPCR analysisof SMN2 RNA. Results are presented as fold change in RNA levels relativeto PBS control, normalized to total SMN2 levels. Each of Tables 19-23represents a different experiment.

TABLE 19 Effect of modified oligonucleotides on human SMN2 RNA splicingin homozygous transgenic mice Compound Dose CORTEX SPINAL CORD No. (μg)exon 7⁺ exon 7⁻ exon 7⁺ exon 7⁻ PBS — 1.0 1.0 1.0 1.0 819735 15 2.4 0.43.3 0.3 1212869 15 2.4 0.4 3.2 0.4 1212870 15 2.1 0.5 2.8 0.4 1212873 152.2 0.4 2.0 0.6 1212874 15 2.1 0.5 2.4 0.6 1212875 15 2.1 0.5 2.3 0.51212880 15 1.7 0.6 2.0 0.6 1212881 15 1.8 0.6 2.3 0.6 1212885 15 2.3 0.42.4 0.5 1212887 15 2.0 0.5 2.2 0.5 1212931 15 2.9 0.2 2.9 0.3 1212936 152.9 0.3 3.3 0.3 1212941 15 3.0 0.1 3.4 0.2

TABLE 20 Effect of modified oligonucleotides on human SMN2 RNA splicingin heterozygous transgenic mice Compound Dose CORTEX SPINAL CORD No.(μg) exon 7⁺ exon 7⁻ exon 7⁺ exon 7⁻ PBS — 1.0 1.0 1.0 1.0 396443 15 2.20.5 2.2 0.7 819735 15 2.7 0.5 3.1 0.5 1287122 15 2.9 0.5 2.3 0.6 128712315 3.0 0.4 3.0 0.4 1287124 15 3.0 0.4 3.2 0.3 1287125 15 3.0 0.4 3.0 0.41287126 15 2.8 0.4 2.8 0.4 1287127 15 2.7 0.5 3.0 0.5 1287128 15 2.6 0.53.2 0.5 1287129 15 2.9 0.4 2.9 0.5 1287130 15 3.7 0.1 3.1 0.5 1287131 152.2 0.6 2.7 0.4 1287132 15 3.2 0.3 2.2 0.6 1287133 15 2.9 0.4 2.8 0.41287728 15 2.8 0.6 3.4 0.3 1287729 15 3.1 0.4 3.0 0.3 1287730 15 3.1 0.32.7 0.4 1287731 15 3.3 0.3 2.8 0.5 1287735 15 2.9 0.5 2.6 0.5 1287738 153.7 0.2 3.2 0.3 1287739 15 3.3 0.4 3.2 0.4 1287743 15 3.6 0.4 3.8 0.41287745 15 3.1 0.5 3.8 0.5

TABLE 21 Effect of modified oligonucleotides on human SMN2 RNA splicingin heterozygous transgenic mice Compound Dose CORTEX SPINAL CORD No.(μg) exon 7⁺ exon 7⁻ exon 7⁺ exon 7⁻ PBS — 1 1 1 1.0 396443 15 1.9 0.61.7 0.7 819735 15 2.3 0.5 1.9 0.6

TABLE 22 Effect of modified oligonucleotides on human SMN2 RNA splicingin heterozygous transgenic mice Compound Dose CORTEX SPINAL CORD No.(μg) exon 7⁺ exon 7⁻ exon 7⁺ exon 7⁻ PBS — 1 1 1 1 1364781 15 2.5 0.62.7 0.4 1364780 15 2.8 0.5 2.6 0.5 1364779 15 2.7 0.5 2.6 0.5 1364778 153 0.5 2.7 0.4

TABLE 23 Effect of modified oligonucleotides on human SMN2 RNA splicingin heterozygous transgenic mice Compound Dose CORTEX SPINAL CORD No.(μg) exon 7⁺ exon 7⁻ exon 7⁺ exon 7⁻ PBS — 1 1 1 1 819735 15 2.8 0.5 2.40.6 1332265 15 2.1 0.7 2.6 0.6 1332269 15 2.5 0.6 2.7 0.5 1332268 15 2.90.5 2.3 0.6 1318756 15 2.2 0.7 2.4 0.6 1333508 15 2 0.6 2.2 0.6 133225115 2.9 0.5 1.9 0.7 1332249 15 2.3 0.7 2.3 0.7

Example 4: Activity of Modified Oligonucleotides Complementary to HumanSMN2 in Transgenic Mice, Single Dose (70 μg)

Activity of modified oligonucleotides was tested in human SMN2transgenic mice essentially as described above in Example 2. Thetransgenic mice were divided into groups of 4 mice each. Each mousereceived a single ICV bolus of 70 μg modified oligonucleotide. A groupof 4 mice received PBS as a negative control. Two weeks post treatment,mice were sacrificed and RNA was extracted from cortical brain tissueand spinal cord for real-time qPCR analysis of SMN2 RNA. Results arepresented as fold change in RNA levels relative to PBS control,normalized to total SMN2 levels.

TABLE 24 Effect of modified oligonucleotides on human SMN2 RNA splicingin homozygous transgenic mice Compound Dose CORTEX SPINAL CORD No. (μg)exon 7⁺ exon 7⁻ exon 7⁺ exon 7⁻ PBS — 1   1   1   1   U12969 70 2.5 0.42.4 0.3 U12970 70 2.7 0.3 2.6 0.3

Example 5: Activity of Modified Oligonucleotides Complementary to HumanSMN2 in Transgenic Mice, Multiple Dose

Activity of selected modified oligonucleotides described above wastested in human SMN2 transgenic mice essentially as described above inExample 2. Comparator Compound No. 396443 was also tested in this assay.The transgenic mice were divided into groups of 4 mice each. Each mousereceived a single ICV bolus of modified oligonucleotide at multipledoses as indicated in the tables below. A group of 4 mice received PBSas a negative control. Two weeks post treatment, mice were sacrificedand RNA was extracted from coronal brain and spinal cord for real-timeqPCR analysis of SMN2 RNA. Results are presented as fold change in RNAlevels relative to PBS control, normalized to total SMN2 levels. ED₅₀for exon inclusion (exon 7⁺) was calculated in GraphPad Prism 7 usingnonlinear regression, 4-parameter dose response curve[Y=Bottom+(Top−Bottom)/(1+(10{circumflex over ( )} logEC50/X){circumflex over ( )}HillSlope)].

TABLE 25 Effect of modified oligonucleotides on human SMN2 RNA splicingin homozygous transgenic mice Dose CORONAL BRAIN ED50 SPINAL CORD ED50Compound No. (μg) exon 7⁺ exon 7⁻ (μg) exon 7⁺ exon 7⁻ (μg) PBS — 1.01.0 1.0 1.0 396443 3 1.4 0.9 32.5 1.3 0.9 22.1 10 1.8 0.8 2.0 0.7 30 2.60.5 2.6 0.4 100 3.5 0.4 3.2 0.3 300 4.2 0.1 3.6 0.2 1263789 3 1.5 0.938.3 1.5 0.8 13.3 10 2.0 0.7 2.2 0.6 30 2.3 0.6 3.0 0.4 100 3.4 0.3 3.40.3 300 3.9 0.1 3.7 0.2 1287717 3 1.3 0.8 38.7 1.3 0.9 20.5 10 1.8 0.71.9 0.7 30 2.4 0.7 2.7 0.5 100 3.5 0.4 3.3 0.3 300 4.1 0.1 3.8 0.21358996 3 1.6 0.9 16.6 1.7 0.8 7.4 10 2.5 0.6 2.6 0.5 30 3.0 0.4 3.5 0.2100 4.0 0.2 3.6 0.2 300 4.0 0.1 3.9 0.1 1287745 3 1.5 0.8 22.8 1.7 0.78.8 10 2.1 0.6 2.4 0.5 30 3.0 0.3 3.3 0.3 100 3.6 0.1 3.5 0.2 300 4.20.1 3.8 0.1

Example 6: Tolerability of Modified Oligonucleotides Complementary toSMN2 in Wild-Type Mice, 3 Hour Study

Modified oligonucleotides described above were tested in wild-typefemale C57/B16 mice to assess tolerability. Wild-type female C57/B16mice each received a single ICV dose of 700 μg of modifiedoligonucleotide listed in the tables below. Comparator Compound No.396443 was also tested in this assay with a dose of 350 μg. ComparatorCompound Nos. 387954, 396442, 443305, and 819735 were also tested inthis assay with a dose of 700 μg. Each treatment group consisted of 4mice. A group of 4 mice received PBS as a negative control for eachexperiment (identified in separate tables below). At 3 hourspost-injection, mice were evaluated according to seven differentcriteria. The criteria are (1) the mouse was bright, alert, andresponsive; (2) the mouse was standing or hunched without stimuli; (3)the mouse showed any movement without stimuli; (4) the mousedemonstrated forward movement after it was lifted; (5) the mousedemonstrated any movement after it was lifted; (6) the mouse respondedto tail pinching; (7) regular breathing. For each of the 7 criteria, amouse was given a subscore of 0 if it met the criteria and 1 if it didnot (the functional observational battery score or FOB). After all 7criteria were evaluated, the scores were summed and averaged within eachtreatment group. The results are presented in the tables below. Each ofTables 26-49 represents a different experiment.

TABLE 26 Tolerability dose scores in mice at 350 μg Compound 3 hr NumberFOB PBS 0   396443 4.0

TABLE 27 Tolerability dose scores in mice at 700 μg Compound 3 hr NumberFOB PBS 0.00 443305 4.75

TABLE 28 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0    396442 2.5  524403  3.25 1210339  1.25 1210340  2.251210341  3.75 1210342 0   1210343 0   1212817 0   1212818 0   12128190   1212820 0   1212821 0   1212822 0   1212823 0   1212824 0   12128251   1212826 0   1212827 0   1212828 0   1212829 0   1212830 0   12128310  

TABLE 29 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00  396442 2.50 1210340 3.50 1212850 0.50 1212851 0.75 12128520.00 1212853 0.00 1212854 0.25 1212855 0.25 1212856 0.00 1212857 0.001212858 0.00 1212859 0.00 1212860 0.75 1212861 1.00 1212863 2.00 12128640.00 1212866 0.75 1212867 0.00 1212868 0.00

TABLE 30 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00  396442 3.25 1212961 0.00 1212963 1.00 1212964 2.00 12129651.25 1212966 1.25 1212968 0.00 1212971 1.00 1212972 3.25 1212973 0.501212974 2.00 1212975 0.50 1212976 1.75

TABLE 31 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00 1212977 0.75 1212978 0.00 1212979 1.75 1212980 1.50 12129810.00 1212982 0.50 1212983 0.75 1212984 2.75 1212985 0.00 1212986 1.001212987 1.75 1212988 4.50 1212989 1.75 1212990 4.50 1212991 1.25 12129923.75

TABLE 32 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00 1212993 7.00 1212994 6.50 1212995 4.25 1212996 3.25 12129974.00 1212998 2.00 1212999 1.00 1213000 1.25 1213001 3.00 1213002 2.001213003 4.00 1213004 3.00 1213005 3.75 1213006 4.00 1213007 4.00 12130083.50

TABLE 33 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00 1212832 0.00 1212833 0.00 1212834 0.00 1212835 0.00 12128360.00 1212837 0.00 1212838 0.00 1212839 0.00 1212840 0.00 1212841 0.001212842 0.00 1212843 0.00 1212844 0.25 1212845 1.00 1212846 0.00 12128470.00 1212848 0.00 1212849 0.00

TABLE 34 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00  396442 1.75 1210339 1.00 1212865 1.00 1212962 0.00 12129670.50 1212969 0.50 1212970 1.25

TABLE 35 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00  819735 2.00 1212869 2.00 1212870 4.75 1212871 1.00 12128730.00 1212874 0.00 1212875 0.00 1212879 3.00 1212880 0.00 1212881 4.001212885 1.00 1212887 2.25 1212931 2.00 1212936 2.00 1212941 1.25

TABLE 36 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00 1263778 0.00 1263781 0.00 1263783 0.00 1263785 1.00 12637870.00 1263789 0.00 1263791 0.00 1263793 0.00 1263795 0.00 1263797 0.001263799 0.00 1263800 0.00 1263802 0.00 1263804 0.00 1263806 0.00 12638081.00 1263810 0.00 1263812 0.00 1263814 1.00 1263816 0.50 1263818 0.001263820 0.00 1263822 0.25 1263824 0.00

TABLE 37 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00 1263826 0.00

TABLE 38 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00  387954 4.00 1287048 0.00 1287049 0.00 1287050 2.00 12870513.25 1287052 3.50 1287053 2.75 1287054 2.00 1287055 3.25 1287056 4.001287057 3.00 1287058 4.00 1287059 4.00 1287060 4.00 1287061 4.00 12870623.50

TABLE 39 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00 1287106 3.50 1287107 4.00 1287108 3.75 1287109 3.25 12871103.00 1287111 4.75 1287112 4.00 1287113 3.50 1287114 3.25 1287115 3.501287116 4.00 1287117 4.25 1287118 3.00 1287119 3.50 1287120 3.75 12871212.75

TABLE 40 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00 1287063 0.00 1287064 0.00 1287065 1.00 1287066 3.75 12870671.00 1287068 2.50 1287069 2.25 1287071 1.00 1287072 3.00 1287073 3.751287074 1.75 1287075 3.50 1287076 2.00

TABLE 41 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00 1287070 2.00 1287701 2.50 1287702 3.75 1287703 3.75 12877054.00 1287706 4.00 1287707 4.00 1287709 4.75 1287710 4.00 1287711 4.751287712 4.00 1287713 4.00 1287714 3.50 1287715 4.00 1287716 4.00 12877173.25

TABLE 42 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00 1287728 1.00 1287729 1.25 1287730 2.00 1287731 2.50 12877323.00 1287733 3.25 1287734 3.00 1287735 0.50 1287736 2.50 1287737 4.001287738 3.00 1287739 2.50 1287740 2.75 1287741 3.75 1287742 3.00 12877432.75 1287744 2.25 1287745 1.00

TABLE 43 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00 1287122 0.00 1287123 0.00 1287124 3.50 1287125 3.00 12871263.00 1287127 0.00 1287128 0.00 1287129 4.00 1287130 2.75 1287131 2.501287132 2.75 1287133 3.25 1287704 3.50 1287708 3.50

TABLE 44 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00 1318748 2.00 1318765 4.00 1318767 4.25 1318770 3.75 13187714.50 1318772 4.25 1318773 4.25 1318774 3.50 1318775 3.75 1318776 3.751318777 4.00 1318778 4.00 1318779 4.00 1318780 4.00 1318781 4.00 13187821.00 1318783 4.00 1318784 2.00

TABLE 45 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00 1318757 4.00 1318758 4.25 1318759 3.75 1318760 3.75 13187614.00 1318762 4.00 1318763 4.00 1318764 3.75 1318766 3.75 1318768 4.001318769 4.00

TABLE 46 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00 1318749 4.25 1318750 2.25 1318751 4.00 1318752 3.75 13187532.25 1318754 3.00 1318755 3.75 1318756 0.00

TABLE 47 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00 1332247 1.75 1332248 0.25 1332249 0.00 1332250 3.75 13322510.00 1332252 3.00 1332263 2.00 1332265 1.50 1332266 1.00 1332267 3.751332268 2.75 1332269 1.25 1332270 2.25 1332271 2.50 1333508 0.00

TABLE 48 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00 1332255 1.00 1332256 2.00 1332257 1.25 1332258 1.25 13322592.25 1332260 2.25 1332261 2.50 1332262 2.00

TABLE 49 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00 1358996 0.00 1364777 2.00 1364778 3.00 1364779 3.50 13647803.50 1364781 5.25 1364782 2.50 1364783 3.50 1364784 3.50

Example 7: Tolerability of Modified Oligonucleotides Complementary toHuman SMN2 in Rats, Long-Term Assessment

In separate studies run under the same conditions, selected modifiedoligonucleotides described above were tested in Sprague Dawley rats toassess long-term tolerability. Comparator Compound Nos. 396442 and819735 were also tested in this assay. Sprague Dawley rats each receiveda single intrathecal (IT) delivered dose of 3 mg of oligonucleotide orPBS. Beginning 1 week post-treatment, each animal was weighed andevaluated weekly by a trained observer for adverse events. Adverseevents were defined as neurological dysfunction not typical inPBS-treated control animals, including, but not limited to: abnormallimb splay, abnormal gait, tremors, abnormal respiration, paralysis, andspasticity. The onset of the adverse event is defined as the weekpost-dosing when the dysfunction was first recorded. If no adverse eventwas achieved, there is no onset (−). The onset of adverse eventstypically correlates with a failure to thrive as defined by a lack ofbody weight gain/maintenance similar to PBS-treated animals Similartolerability assessments were described in Oestergaard et al., NucleicAcids Res., 2013 November, 41(21), 9634-9650 and Southwell et al., MolTher., 2014 Dec. 22(12), 2093-2106.

At the end of the study, the rats were sacrificed and tissues werecollected. Histopathology was performed on sections of cerebellum usingcalbindin stain. Purkinje cell loss was observed in calbindin stainedcerebellum sections as indicated in the table below. Cerebellum andspinal cord were also evaluated using an antibody specific for modifiedoligonucleotides. Animals demonstrating no oligonucleotide uptake wereexcluded from histopathology analysis. Histology was not completed foranimals that were sacrificed early due to adverse events. Additionally,cortical GFAP, a marker of astrogliosis (Abdelhak, et al., ScientificReports, 2018, 8, 14798), was measured using RT-PCR, and averageelevations>2-fold are noted below.

TABLE 50 Long-term tolerability in rats at 3 mg dose Adverse Purkinjeevent cell onset, loss (# Cortex weeks animals GFAP post- with mRNAtreatment, loss/# >2-fold Compound individual animals PBS Number animalstested) Control PBS No Not observed N/A  396442 6, 6, 2 2/3 Yes  8197354, 6, 6, — 1/4 Yes 1263789 —, —, — 0/3 No 1287717 —, —, —, —, —, —, —, —0/8 No 1287745 —, —, —, —, —, —, — 0/7 No 1358996 —, —, —, — 0/4 No1263783 —, —, —, — 0/4 No 1263785 —, —, — 0/3 No 1263787 —, —, —, — 0/4No 1263800 —, — 0/2 No 1263802 —, —, — 0/3 No 1263806 —, —, — 0/3 No1263808 —, —, — 0/3 No 1263810 —, —, — 0/3 No

Example 8: Tolerability and Pharmacokinetics of ModifiedOligonucleotides in Non-Human Primates, Single or Repeat Dosing

Cynomolgus monkeys are treated with modified oligonucleotides todetermine the local and systemic tolerability and pharmacokinetics ofthe modified oligonucleotides. Each group receives either artificial CSFor modified oligonucleotide as a single intrathecal lumbar bolus doseinjection (IT), or, for repeat-dosing groups, an IT bolus dose on day 1of the study, followed by IT bolus doses at later time points. Tissuesare collected 1 week after the final injection.

In a single dose study, monkeys are administered a single dose ofmodified oligonucleotide and tolerability is assessed. Representativedoses for single-dose studies in adult cynomolgus monkeys include 1 mg,3 mg, 7 mg, and 35 mg.

In a repeat-dosing study, monkeys are administered an IT bolus dose onday 1 of the study, followed by weekly (e.g., days 8, 15, and 22 for afour-week study) or monthly (e.g., days 29, 57, and 84 for a 13 weekstudy) IT bolus dosing. Representative doses for repeat-dose studies inadult cynomolgus studies include 1 mg, 3 mg, 7 mg, and 35 mg. Assessmentof tolerability is based on clinical observations, body weights, foodconsumption, physical and neurological examinations includingsensorimotor reflexes, cerebral reflexes and spinal reflexes,coagulation, hematology, clinical chemistry (blood and cerebral spinalfluid (CSF)), cell count, and anatomic pathology evaluations. Completenecropsies are performed with a recording of any macroscopicabnormality. Organ weights are taken and microscopic examinations areconducted. Blood is collected for complement analysis. In addition,blood, CSF, and tissues (at necropsy) are collected for toxicokineticevaluations.

Tolerability of modified oligonucleotides is analyzed in brain andspinal cord tissue by measuring Aif1 and Gfap levels in cynomolgusmonkeys treated with the modified oligonucleotide or the control. Brainand spinal cord samples are collected and flash frozen in liquidnitrogen and stored frozen (−60° C. to −90° C.). At time of sampling, 2mm biopsy punches are used to collect samples from frozen tissues forRNA analysis. Punches are taken from multiple brain and spinal cordregions.

Example 9: Phase Ia Human Clinical Trial with Compound No. 1263789,1287717, 1287745, or 1358996

Safety, tolerability, pharmacokinetics, pharmacodynamics and efficacy ofmodified oligonucleotide complementary to human SMN2 is evaluated in aclinical trial setting. Single and/or multiple doses of modifiedoligonucleotide are evaluated in patients with confirmed SMA, such asType I SMA, Type II SMA, Type III SMA, or Type IV SMA.

Patient safety is monitored closely during the study. Safety andtolerability evaluations include: physical examination and standardneurological assessment (including fundi), vital signs (HR, BP,orthostatic changes, weight), ECG, AEs and concomitant medications,Columbia Suicide Severity Rating Scale (C-SSRS), CSF safety labs (cellcounts, protein, glucose), plasma laboratory tests (clinical chemistry,hematology), and urinalysis.

Efficacy evaluations are selected that are age and Type appropriate andinclude, for example, the Hammersmith Motor Function Scale—Expanded(HFMSE), which is a reliable and validated tool used to assess motorfunction in children with SMA; the Pediatric Quality of Life Inventory(PedsQL™) Measurement 4.0 Generic Core Scale; the Pediatric Quality ofLife Inventory 3.0 Neuromuscular Modules; the Compound Muscle ActionPotential (CMAP); the Motor Unit Number Estimation (MUNE); the UpperLimb Module (ULM); and the 6-Minute Walk Test (6MWT) (Darras, et al.,Neurology, 2019, 92: e2492-e2506).

Example 10: Design of Modified Oligonucleotides Complementary to a HumanSMN2 Nucleic Acid

Modified oligonucleotides complementary to a human SMN2 nucleic acidwere designed and synthesized as indicated in the tables below.

Each modified oligonucleotide listed in the tables below is 100%complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14truncated from nucleotides 19939708 to Ser. No. 19/967,777). “Startsite” indicates the 5′-most nucleoside to which the modifiedoligonucleotide is complementary in the target nucleic acid sequence.“Stop site” indicates the 3′-most nucleoside to which the modifiedoligonucleotide is complementary in the target nucleic acid sequence.

The modified oligonucleotides in the table below are 18 nucleosides inlength. Each nucleoside comprises either a 2′-MOE sugar moiety or a2′-NMA sugar moiety. The sugar motif for each modified oligonucleotideis provided in the Sugar Motif column, wherein each ‘e’ represents a2′-MOE sugar moiety, and each ‘n’ represents a 2′-NMA sugar moiety. Eachinternucleoside linkage is either a phosphorothioate internucleosidelinkage, a phosphodiester internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, or a mesyl phosphoramidate (MsP)internucleoside linkage. The internucleoside linkage motif for eachmodified oligonucleotide, is provided in the Internucleoside LinkageMotif column, wherein each ‘s’ represents a phosphorothioateinternucleoside linkage, each ‘o’ represents a phosphodiesterinternucleoside linkage, each ‘x’ represents a methoxypropyl phosphonateinternucleoside linkage, and each ‘z’ represents a mesyl phosphoramidate(MsP) internucleoside linkage. Each cytosine is a 5-methyl cytosine.Modified oligonucleotide 449320 has been previously described inWO2015/161170 A2.

TABLE 51 MOE and NMA modified oligonucleotides with mixed PO/PS, PO/MsP,uniform MsP, or PS/MOP internucleoside linkages SEQ ID SEQ IDInternucleoside No: 1 No: 1 SEQ Compound Sugar Motif Linkage Motif StartStop ID Number Sequence (5′ to 3′) (5′ to 3′) (5′ to 3′) Site Site No.449320 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee ssoooooooooooooss 2706227079 23 1287723 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnenn sssssssssssssssxs27062 27079 23 1287724 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnenssssssssssssssssx 27062 27079 23 1287727 CACTTTCATAATGCTGGCnnnnnnnnnnnnnnnnen ssssssssssssssssx 27061 27078 21 1405549TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee zzzzzzzzzzzzzzzzz 27062 27079 231405552 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee ssssssssssszzzzzz 2706227079 23 1405553 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee ssssszzzzzzssssss27062 27079 23 1545359 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnssoooooooooooooss 27062 27079 23 1547773 TCACTTTCATAATGCTGGeeeeeeeeeeeeeeeeee zzooooooooooooozz 27062 27079 23 1549028TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee zzzzooooooooooozz 27062 27079 231549029 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee zzzzzzooooooooozz 2706227079 23 1549030 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee zzzzzzzzooooooozz27062 27079 23

The modified oligonucleotides in the table below all consist of thesequence (from 5′ to 3′): TCACTTTCATAATGCTGG (SEQ ID NO: 23). Eachmodified oligonucleotide listed in the tables below is 100%complementary to SEQ ID NO: 1 (described herein above). “Start site”indicates the 5′-most nucleoside to which the modified oligonucleotideis complementary in the target nucleic acid sequence. “Stop site”indicates the 3′-most nucleoside to which the modified oligonucleotideis complementary in the target nucleic acid sequence. The modifiedoligonucleotides in the table below are 18 nucleosides in length. Eachnucleoside comprises either a 2′-MOE sugar moiety or a 2′-NMA sugarmoiety. The sugar motif for each modified oligonucleotide is provided inthe Sugar Motif column, wherein each ‘e’ represents a 2′-MOE sugarmoiety, and each ‘n’ represents a 2′-NMA sugar moiety. Eachinternucleoside linkage is either a phosphorothioate internucleosidelinkage, a phosphodiester internucleoside linkage, or a mesylphosphoramidate (MsP) internucleoside linkage. The internucleosidelinkage motif for each modified oligonucleotide, is provided in theInternucleoside Linkage Motif column, wherein each ‘s’ represents aphosphorothioate internucleoside linkage, each ‘o’ represents aphosphodiester internucleoside linkage, and each ‘z’ represents a mesylphosphoramidate (MsP) internucleoside linkage. Each cytosine is a5-methyl cytosine. The modified oligonucleotides in the table below areconjugated to a 6-palmitamidohexyl phosphate conjugate group attached tothe 5′-OH of the oligonucleotide. The structure for the conjugate groupis:

TABLE 52 6-palmitamidohexyl conjugated MOE and NMA modifiedoligonucleotides with mixed PO/PS, PO/MsP,or uniform MsP internucleoside linkages SEQ ID SEQ ID InternucleosideNo: 1 No: 1 Compound Sugar Motif Linkage Motif Start Stop SEQ ID Number(5′ to 3′) (5′ to 3′) Site Site No. 1545361 eeeeeeeeeeeeeeeeeessoooooooooooooss 27062 27079 23 1545362 nnnnnnnnnnnnnnnnnnssoooooooooooooss 27062 27079 23 1547772 eeeeeeeeeeeeeeeeeezzzzzzzzzzzzzzzzz 27062 27079 23 1547774 eeeeeeeeeeeeeeeeeezzooooooooooooozz 27062 27079 23 1549031 eeeeeeeeeeeeeeeeeezzzzooooooooooozz 27062 27079 23 1549032 eeeeeeeeeeeeeeeeeezzzzzzooooooooozz 27062 27079 23 1549033 eeeeeeeeeeeeeeeeeezzzzzzzzooooooozz 27062 27079 23The modified oligonucleotides in the table below all consist of thesequence (from 5′ to 3′): TCACTTTCATAATGCTGG (SEQ ID NO: 23), with astart site of 27062, and a stop site of 27079 on SEQ ID No: 1 (describedherein above), wherein “start site” indicates the 5′-most nucleoside towhich the modified oligonucleotide is complementary in the targetnucleic acid sequence, and wherein “stop site” indicates the 3′-mostnucleoside to which the modified oligonucleotide is complementary in thetarget nucleic acid sequence.

The modified oligonucleotides in the table below are 18 nucleosides inlength. The sugar and internucleoside linkage motifs for each modifiedoligonucleotide are provided in the Sequence and Chemistry Notationcolumn, wherein each subscript ‘n’ represents a 2′-NMA sugar moiety,each subscript [DMA]′ represents a 2′-O—(N,N-dimethyl) acetamide moiety,each subscript [NEA]′ represents a 2′-O—(N-ethyl) acetamide moiety, eachsubscript [NPA]′ represents a 2′-O—(N-propyl) acetamide moiety, eachsubscript [NcPA]′ represents a 2′O—(N-cyclopropyl) acetamide moiety,each subscript [McPA]′ represents a 2′-O—(N-cyclopropylmethyl) acetamidemoiety, and each subscript ‘s’ represents a phosphorothioateinternucleoside linkage. Each cytosine is a 5-methyl cytosine, whereinthe superscript ‘m’ before the cytosine residue (NC) represents a5-methyl cytosine. The structures for each of the sugars represented inthe table below are:

TABLE 53NMA and NMA analog modified oligonucleotides with uniform PS internucleoside linkagesCompound SEQ Number Sequence and Chemistry Notation (5′ to 3′) ID No.1355763 T_([DMA]s) ^(m)C_([DMA]s)A_(ns)^(m)C_([DMA]s)T_([DMA]s)T_([DMA]s)^(m)C_([DMA]s)A_(ns)T_([DMA]s)A_(ns)A_(ns)T_([DMA]s)G_(ns)^(m)C_([DMA]s)T_([DMA]s)G_(ns)G_(n) 23 1359463 T_([NEA]s)^(m)C_([NEA]s)A_(ns) ^(m)C_([NEA]s)T_([NEA]s)T_([NEA]s)T_([NEA]s)^(m)C_([NEA]s)A_(ns)T_([NEA]s)A_(ns)A_(ns)T_([NEA]s)G_(ns)^(m)C_([NEA]s)T_([NEA]s)G_(ns)G_(n) 23 1358995 T_([NPA]s)^(m)C_([NPA]s)A_(ns) ^(m)C_([NPA]s)T_([NPA]s)T_([NPA]s)T_([NPA]s)^(m)C_([NPA]s)A_(ns)T_([NPA]s)A_(ns)A_(ns)T_([NPA]s)G_(ns)^(m)C_([NpA]s)T_([NpA]s)G_(ns)G_(n) 23 1355776 T_([NcPA]s)^(m)C_([NcPA]s)A_(ns) ^(m)C_([NcPA]s)T_([NcPA]s)T_([NcPA]s)T_([NcPA]s)^(m)C_([NcPA]s)A_(ns)T_([NcPA]s)A_(ns)A_(ns)T_([NcPA]s)G_(ns)^(m)C_([NcPA]s)T_([NcPA]s)G_(ns)G_(n) 23 1355777 T_([McPA]s)^(m)C_([McPA]s)A_(ns) ^(m)C_([McPA]s)T_([McPA]s)T_([McPA]s)T_([McPA]s)^(m)C_([McPA]s)A_(ns)T_([McPA]s)A_(ns)A_(ns)T_([McPA]s)G_(ns)^(m)C_([McPA]s)T_([McPA]s)G_(ns)G_(n) 23

Example 11: Activity of Modified Oligonucleotides Complementary to HumanSMN2 in Transgenic Mice, Single Dose (35 μg)

Activity of selected modified oligonucleotides described above wastested in human SMN2 transgenic mice essentially as described above inExample 2.

Treatment

The transgenic mice were divided into groups of 4 mice each. Each mousereceived a single ICV bolus of modified oligonucleotide at doses asindicated in the tables below. A group of 4 mice received PBS as anegative control. Two weeks post treatment, mice were sacrificed and RNAwas extracted from coronal brain and spinal cord for real-time qPCRanalysis of SMN2 RNA. Results are presented as fold change in RNA levelsrelative to PBS control, normalized to total SMN2 levels. ED₅₀ for exoninclusion (exon 7⁺) was calculated in GraphPad Prism 7 using nonlinearregression, 4-parameter dose response curve[Y=Bottom+(Top−Bottom)/(1+(10{circumflex over ( )} logEC50/X){circumflex over ( )}HillSlope)].

RNA Analysis

Two weeks post treatment, mice were sacrificed and RNA was extractedfrom cortical brain tissue and spinal cord for real-time qPCR analysisof SMN2 RNA. Primer probe set hSMN2vd#4_LTS00216_MGB was used todetermine the amount of SMN2 RNA including exon 7 (exon 7⁺). Primerprobe set hSMN2_Sumner68_PPS50481 was used to determine the amount ofSMN2 RNA excluding exon 7 (exon 7⁻). Total SMN2 RNA levels were measuredusing primer probe set hSMN2_LTS00935. Results are presented as foldchange in RNA levels relative to PBS control, normalized to total SMN2levels.

TABLE 54 Effect of modified oligonucleotides on human SMN2 RNA splicingin heterozygous transgenic mice Compound Dose CORTEX SPINAL CORD No.(μg) exon 7⁺ exon 7⁻ exon 7⁺ exon 7⁻ PBS — 1   1   1   1    396443 353   0.6 3   0.5 1405549 35 1.6 0.9 1.2 0.9 1405552 35 3.2 0.6 2.1 0.71405553 35 2.4 0.7 1.7 0.8

TABLE 55 Effect of modified oligonucleotides on human SMN2 RNA splicingin heterozygous transgenic mice Compound Dose CORTEX SPINAL CORD No.(μg) exon 7⁺ exon 7⁻ exon 7⁺ exon 7⁻ PBS — 1   1   1   N/A  396443 352.7 0.5 3   N/A 1405549 35 1.5 0.8 1.9 N/A 1547772 35 1.8 0.8 1.8 N/A1547773 35 1.6 0.9 1.9 N/A 1547774 35 1.6 1   1.6 N/A 1549028 35 1.5 1  1.5 N/A 1549029 35 1.4 1   1.7 N/A 1549030 35 1.4 1   1.4 N/A 1549031 351.7 0.8 1.6 N/A 1549032 35  1.4†  0.9†  1.7† N/A 1549033 35 1.4 1   1.3N/A †indicates fewer than four samples available

Example 12: Activity of Modified Oligonucleotides Complementary to HumanSMN2 in Transgenic Mice, Single Dose (15 μg)

Activity of selected modified oligonucleotides described above wastested in human SMN2 transgenic mice essentially as described above inExample 2.

Treatment

The transgenic mice were divided into groups of 4 mice each. Each mousereceived a single ICV bolus of modified oligonucleotide at doses asindicated in the tables below. A group of 4 mice received PBS as anegative control. Two weeks post treatment, mice were sacrificed and RNAwas extracted from coronal brain and spinal cord for real-time qPCRanalysis of SMN2 RNA. Results are presented as fold change in RNA levelsrelative to PBS control, normalized to total SMN2 levels. ED₅₀ for exoninclusion (exon 7⁺) was calculated in GraphPad Prism 7 using nonlinearregression, 4-parameter dose response curve[Y=Bottom+(Top−Bottom)/(1+(10{circumflex over ( )} logEC50/X){circumflex over ( )}1-HillSlope)].

RNA Analysis

Two weeks post treatment, mice were sacrificed and RNA was extractedfrom cortical brain tissue and spinal cord for real-time qPCR analysisof SMN2 RNA. Primer probe set hSMN2vd#4_LTS00216_MGB was used todetermine the amount of SMN2 RNA including exon 7 (exon 7⁺). Primerprobe set hSMN2_Sumner68_PPS50481 was used to determine the amount ofSMN2 RNA excluding exon 7 (exon 7⁻). Total SMN2 RNA levels were measuredusing primer probe set hSMN2_LTS00935. Results are presented as foldchange in RNA levels relative to PBS control, normalized to total SMN2levels.

TABLE 56 Effect of modified oligonucleotides on human SMN2 RNA splicingin heterozygous transgenic mice Compound Dose CORTEX SPINAL CORD No.(μg) exon 7⁺ exon 7⁻ exon 7⁺ exon 7⁻ PBS — 1   1   1   1    443305 153.4 0.4 3.3 0.3 1287723 15 3.9 0.3 3.3 0.3 1287724 15 4.2 0.2 3.7 0.21287727 15 4.5 0.2 3.4 0.2

Example 13: Activity of Modified Oligonucleotides Complementary to HumanSMN2 in Transgenic Mice, Multiple Dose

Activity of selected modified oligonucleotides described above wastested in human SMN2 transgenic mice essentially as described above inExample 2.

Treatment

The transgenic mice were divided into groups of 4 mice each. Each mousereceived a single ICV bolus of modified oligonucleotide at multipledoses as indicated in the tables below. A group of 4 mice received PBSas a negative control. Two weeks post treatment, mice were sacrificedand RNA was extracted from coronal brain and spinal cord for real-timeqPCR analysis of SMN2 RNA. Results are presented as fold change in RNAlevels relative to PBS control, normalized to total SMN2 levels. ED₅₀for exon inclusion (exon 7⁺) was calculated in GraphPad Prism 7 usingnonlinear regression, 4-parameter dose response curve[Y=Bottom+(Top−Bottom)/(1+(10{circumflex over ( )} logEC50/X){circumflex over ( )}1-HillSlope)].

RNA Analysis

Two weeks post treatment, mice were sacrificed and RNA was extractedfrom cortical brain tissue and spinal cord for real-time qPCR analysisof SMN2 RNA. Primer probe set hSMN2vd#4_LTS00216_MGB was used todetermine the amount of SMN2 RNA including exon 7 (exon 7⁺). Primerprobe set hSMN2_Sumner68_PPS50481 was used to determine the amount ofSMN2 RNA excluding exon 7 (exon 7⁻). Total SMN2 RNA levels were measuredusing primer probe set hSMN2_LTS00935. Results are presented as foldchange in RNA levels relative to PBS control, normalized to total SMN2levels.

TABLE 57 Effect of modified oligonucleotides on human SMN2 RNA splicingin heterozygous transgenic mice Dose CORTEX ED50 SPINAL CORD ED50Compound No. (μg) exon 7⁺ exon 7⁻ (μg) exon 7⁺ exon 7⁻ (μg) PBS — 1 1 —1 1 — 396443 10 2.1 0.7 26 1.9 0.8 22 30 2.8 0.5 2.6 0.7 100 3.2 0.3 2.80.4 449320 10 1.2 1.0 >100 1.2 1.2 >100 30 1.5 1.0 1.3 1.2 100 1.5 0.91.3 0.9 1545361 10 1.4 1.0 >100 1.2 1.1 >100 30 1.8 1.1 1.6 1.3 100 1.30.9 1.4 0.9 443305 10 2.4 0.6 14 2.6 0.5 9 30 3.5 0.3 3.0 0.4 100 3.70.1 3.3 0.1 1545359 10 1.4 1.0 >100 1.4 1.1 >100 30 2.0 0.9 1.7 1.1 1002.3 0.6 1.7 0.8 1545362 10 1.4 0.9 95 1.3 1.1 51 30 1.9 0.8 2.4 1.0 1002.7 0.5 2.6 0.6

TABLE 58 Effect of modified oligonucleotides on human SMN2 RNA splicingin heterozygous transgenic mice Dose CORTEX ED50 SPINAL CORD ED50Compound No. (μg) exon 7⁺ exon 7⁻ (μg) exon 7⁺ exon 7⁻ (μg) PBS — 1 1 —1   1   — 1263789 3 1.5 0.9 35 1.6 0.8 16 10 1.9 0.8 2.2 0.6 30 2.7 0.62.9 0.5 100 3.8 0.3 3.5 0.3 300 4.3 0.2 3.9 0.2 1287703 3 1.5 0.7 43 1.50.8 28 10 1.6 0.7 2.0 0.7 30 2.7 0.5 2.6 0.5 100 3.7 0.3 3.2 0.4 300 4.00.2 3.5 0.2 1287717 3 1.4 0.8 31 1.4 0.9 30 10 1.7 0.7 1.8 0.7 30 3.10.4  2.4†  0.5† 100 3.8 0.3 3.3 0.3 300 4.3 0.2 3.8 0.2 1318768 3 1.30.8 49 1.2 0.8 32 10 1.8 0.7 1.8 0.8 30 2.4 0.6 2.4 0.6 100 3.5 0.3 3.30.4 300 4.1 0.2 3.9 0.2 1287731 3 1.5 0.8 29 1.5 1.0 13 10 2.0 0.6 2.40.6 30 2.7 0.4 3.2 0.4 100 4.3 0.2 3.8 0.2 300 4.2 0.1 3.7 0.1 1287735 31.7 0.7 22 1.5 0.8 15 10 2.4 0.6 2.2 0.6 30 3.0 0.3 3.1 0.3 100 4.0 0.23.5 0.2 300 4.2 0.1 3.9 0.1 1287745 3 1.5 0.7 35 1.4 0.8 13 10 2.0 0.62.4 0.6 30 2.9 0.4 3.2 0.4 100 3.7 0.2 3.6 0.2 300 3.7 0.2 3.8 0.2396443 3 1.7 0.7 47 1.5 0.9 22 10 1.4 0.7 1.6 0.8 30 2.8 0.5 3.1 0.4 1003.3 0.4 3.3 0.5 300 4.3 0.2 4.0 0.2 † indicates fewer than four samplesavailable

TABLE 59 Effect of modified oligonucleotides on human SMN2 RNA splicingin heterozygous transgenic mice Dose CORTEX ED50 SPINAL CORD ED50Compound No. (μg) exon 7⁺ exon 7⁻ (μg) exon 7⁺ exon 7⁻ (μg) PBS — 1 1 —1 1 — 396443 3 1.1 0.9 39 1.2 0.9 33 30 2.0 0.6 2.3 0.5 100 2.3 0.4 2.70.3 443305 3 1.4 0.7 13 1.6 0.7 10 30 2.4 0.3 3.0 0.2 100 2.9 0.2 3.10.2 1355763 3 1.1 0.8 54 1.1 0.8 45 30 1.7 0.6 2.1 0.5 100 2.4 0.4 2.60.3 1359463 3 1.3 0.8 18 1.3 0.7 19 30 2.3 0.3 2.7 0.3 100 2.9 0.2 2.70.2 1358995 3 1.2 0.8 45 1.0 0.8 59 30 2.0 0.4 1.8 0.4 100 2.2 0.4 2.60.3 1355776 3 1.1 0.8 25 1.3 0.7 24 30 2.2 0.4 2.4 0.4 100 2.6 0.3 2.90.3 1355777 3 1.0 0.9 107 0.9 0.9 72 30 1.6 0.7 1.8 0.6 100 1.7 0.5 2.20.6

Example 15: Tolerability of Modified Oligonucleotides Complementary toSMN2 in Wild-Type Mice

Modified oligonucleotides described above were tested in wild-typefemale C57/B16 mice to assess the tolerability of the oligonucleotides.Wild-type female C57/B16 mice each received a single ICV dose of 700 μgof modified oligonucleotide listed in the table below. Each treatmentgroup consisted of 4 mice. A group of 4 mice received PBS as a negativecontrol for each experiment (identified in separate tables below). At 3hours post-injection, mice were evaluated according to seven differentcriteria. The criteria are (1) the mouse was bright, alert, andresponsive; (2) the mouse was standing or hunched without stimuli; (3)the mouse showed any movement without stimuli; (4) the mousedemonstrated forward movement after it was lifted; (5) the mousedemonstrated any movement after it was lifted; (6) the mouse respondedto tail pinching; (7) regular breathing. For each of the 7 criteria, amouse was given a subscore of 0 if it met the criteria and 1 if it didnot (the functional observational battery score or FOB). After all 7criteria were evaluated, the scores were summed for each mouse andaveraged within each treatment group. The results are presented in thetables below.

TABLE 60 Tolerability scores in mice at 700 μg dose Compound 3 hr NumberFOB PBS 0.00 1287723 2.00 1287724 1.00 1287727 2.00

1. A modified oligonucleotide according to the following chemicalstructure:

or a salt thereof.
 2. The modified oligonucleotide of claim 1, which isthe sodium salt or the potassium salt.
 3. A modified oligonucleotideaccording to the following chemical structure:


4. A chirally enriched population of modified oligonucleotides of claim1, wherein the population is enriched for modified oligonucleotidescomprising at least one particular phosphorothioate internucleosidelinkage having a particular stereochemical configuration.
 5. Thechirally enriched population of claim 1, wherein the population isenriched for modified oligonucleotides comprising at least oneparticular phosphorothioate internucleoside linkage having the (Sp)configuration.
 6. The chirally enriched population of claim 1, whereinthe population is enriched for modified oligonucleotides comprising atleast one particular phosphorothioate internucleoside linkage having the(Rp) configuration.
 7. The chirally enriched population of claim 1,wherein the population is enriched for modified oligonucleotides havinga particular, independently selected stereochemical configuration ateach phosphorothioate internucleoside linkage.
 8. The chirally enrichedpopulation of claim 1, wherein the population is enriched for modifiedoligonucleotides having the (Sp) configuration at each phosphorothioateinternucleoside linkage or for modified oligonucleotides having the (Rp)configuration at each phosphorothioate internucleoside linkage.
 9. Thechirally enriched population of claim 1, wherein the population isenriched for modified oligonucleotides having the (Rp) configuration atone particular phosphorothioate internucleoside linkage and the (Sp)configuration at each of the remaining phosphorothioate internucleosidelinkages.
 10. The chirally enriched population of claim 1, wherein thepopulation is enriched for modified oligonucleotides having at least 3contiguous phosphorothioate internucleoside linkages in the Sp, Sp, andRp configurations, in the 5′ to 3′ direction.
 11. A population ofmodified oligonucleotides of claim 1, wherein all of thephosphorothioate internucleoside linkages of the modifiedoligonucleotide are stereorandom.
 12. An oligomeric compound comprisinga modified oligonucleotide according to the following chemical notation:^(m)C_(ns) A_(no) ^(m)C_(ns) T_(no) T_(ns) T_(ns) ^(m)C_(ns) A_(ns)T_(ns) A_(ns) A_(ns) T_(ns) G_(ns) ^(m)C_(ns) T_(ns) G_(ns) G_(ns)^(m)C_(n) (SEQ ID NO: 21), wherein: A=an adenine nucleobase, ^(m)C=a5-methyl cytosine nucleobase, G=a guanine nucleobase, T=a thyminenucleobase, n=a 2′-NMA sugar moiety, s=a phosphorothioateinternucleoside linkage, and o=a phosphodiester internucleoside linkage.13. The oligomeric compound of claim 12, wherein the modifiedoligonucleotide is linked to a conjugate group.
 14. A pharmaceuticalcomposition comprising the modified oligonucleotide of claim 1 and apharmaceutically acceptable diluent or carrier.
 15. The pharmaceuticalcomposition of claim 14, wherein the pharmaceutically acceptable diluentis artificial CSF (aCSF) or PBS.
 16. The pharmaceutical composition ofclaim 15, wherein the pharmaceutical composition consists essentially ofthe modified oligonucleotide and artificial CSF (aCSF) or PBS.
 17. Apharmaceutical composition comprising the modified oligonucleotide ofclaim 3 and a pharmaceutically acceptable diluent or carrier.
 18. Thepharmaceutical composition of claim 17, wherein the pharmaceuticallyacceptable diluent is artificial CSF (aCSF) or PBS.
 19. Thepharmaceutical composition of claim 18, wherein the pharmaceuticalcomposition consists essentially of the modified oligonucleotide andartificial CSF (aCSF) or PBS.
 20. A pharmaceutical compositioncomprising the oligomeric compound of claim 12 and a pharmaceuticallyacceptable diluent or carrier.
 21. The pharmaceutical composition ofclaim 20, wherein the pharmaceutically acceptable diluent is artificialCSF (aCSF) or PBS.
 22. The pharmaceutical composition of claim 21,wherein the pharmaceutical composition consists essentially of theoligomeric compound and artificial CSF (aCSF) or PBS.
 23. Apharmaceutical composition comprising the population of modifiedoligonucleotides of claim 4, and a pharmaceutically acceptable diluentor carrier.
 24. The pharmaceutical composition of claim 23, wherein thewherein the pharmaceutically acceptable diluent is artificial CSF (aCSF)or PBS.
 25. The pharmaceutical composition of claim 24, wherein thepharmaceutical composition consists essentially of the population ofmodified oligonucleotides and artificial CSF (aCSF) or PBS.
 26. A methodof treating a disease associated with SMN1 or SMN2 comprisingadministering to a subject having or at risk for developing a diseaseassociated with SMN1 or SMN2 a therapeutically effective amount of apharmaceutical composition according to claim 14; thereby treating thedisease associated with SMN1 or SMN2.
 27. A method of treating a diseaseassociated with SMN1 or SMN2 comprising administering to a subjecthaving or at risk for developing a disease associated with SMN1 or SMN2a therapeutically effective amount of a pharmaceutical compositionaccording to claim 17; thereby treating the disease associated with SMN1or SMN2.
 28. A method of treating a disease associated with SMN1 or SMN2comprising administering to a subject having or at risk for developing adisease associated with SMN1 or SMN2 a therapeutically effective amountof a pharmaceutical composition according to claim 20; thereby treatingthe disease associated with SMN1 or SMN2.
 29. The method of claim 26,wherein the disease is any of Type I SMA, Type II SMA, Type III SMA, orType IV SMA.
 30. The method of claim 29, wherein at least one symptom ofSMA is ameliorated.
 31. The method of claim 30, wherein the symptom isany of reduced muscle strength; inability or reduced ability to situpright, to stand, and/or walk; reduced neuromuscular activity; reducedelectrical activity in one or more muscles; reduced respiration;inability or reduced ability to eat, drink, and/or breathe withoutassistance; loss of weight or reduced weight gain; and/or decreasedsurvival.